MEDICAL    .SCHOOL 


BACTERIOLOGICAL    TECHNIQUE    AND 
SPECIAL   BACTERIOLOGY 


a 


M  A^N  UAL 


OF 


BACTERIOLOGICAL  TECHNIQUE 


AND 


SPECIAL  BACTERIOLOGY 


BY 

THOMAS  JSOWHILL,   F.R.C.V.S.,  F.R.P.S. 

Late  Special  Lecturer  on  Bacteriology, Medical  Department  and  Professor  of  Bacteriology, 

Veterinary  Department,  University  of  California  ;  Late  Special  Agent,  Bureau 

of  Animal  Industry,   Washington,  D.C.;  Late  Veterinary  Inspector  for 

the  City  and  County  of  San  Francisco,  California,  etc.,  etc. 


WITH 
ONE  HUNDRED  ORIGINAL  ILLUSTRATIONS 


NEW  YORK, 
WILLIAM  WOOD  &  COMPANY, 

MDCCCXCIX 


PREFACE 

THIS  Manual  of  Bacteriological  Technique  and  Special  Bacteriology  has 
been  written  in  pursuance  of  a  plan  first  adopted  by  the  writer  while 
working  in  the  Hygienic  Institute,  Berlin,  and  is  the  result  of  consider- 
able experience  of  the  wants  of  students  in  Bacteriology,  both  here  and 
abroad.  The  technique  and  working  methods  have  been  carefully 
selected,  and  from  the  mass  of  available  material  on  this  rapidly  growing 
branch  of  the  subject,  only  those  methods  and  material  have  been 
chosen  which  possess  distinctive  benefits.  The  methods  are  all  straight- 
forward and  practical,  and  when  carefully  performed  give  excellent 
results. 

It  was  thought  best  to  separate  the  Technique  from  the  Bacteriology 
of  Special  Diseases,  which  latter  is  included  in  the  Special  Bacteriology. 
This  has  been  made  very  inclusive,  and  it  is  thought  that  it  is  quite 
sufficiently  so,  for  any  one  not  so  specially  equipped  as  to  go  to  the 
original  papers  and  articles.  Much  that  is  of  importance  in  the  study  of 
the  comparative  diseases  of  animals  and  those  transmissible  to  man  has 
been  included,  an  inclusion  very  necessary  in  the  light  of  the  growing 
importance  of  this  to  the  practical  hygienist.  Again,  to  meet  the  needs 
of  Sanitarians  an  account  has  been  given  of  the  common  bacteria  found 
in  water,  milk,  air,  soil,  etc.  It  is  generally  agreed  that  a  book  of  this 
description  is  not  a  fitting  place  for  original  or  controversial  work,  and 
that  the  object  to  be  attained  is  the  presenting  of  a  correct  idea  of  the 
relative  conditions  of  the  contemporary  science — consequently  little  is 
alluded  to  of  the  above  nature,  and  nothing  speculative  which  could  be 
avoided  without  interfering  with  inevitable  deductions  from  fundamental 
experimental  principles. 

I    take    the   opportunity    of  expressing   my  thanks   to    Dr    George 
Nuttall  of  the  Hygienic  Institute,  Berlin,  for  many  valuable  suggestions, 


a 


13557 


vi  PREFACE 

and  for  revising  the  manuscript  of  the  technical  portion  of  this  work  ; 
to  Professor  Giinther  of  the  same  Institute  for  the  many  pure  cultures 
he  provided  me  with  ;  to  Herr  Hansel,  Manager  for  Carl  Zeiss  in 
Berlin,  for  his  assistance  in  making  the  negatives  reproduced  by  the 
colotype  process  in  the  four  plates  at  the  end  of  this  work,  also  for 
valuable  assistance  in  connection  with  the  other  illustrations ;  to 
Dr  M'Lean,  Fortrose;  N.B.,  for  revising  the  proof  sheets  and  valuable 
suggestions  regarding  same. 

I  am  also  indebted  to  Herr  Paul  Altaian,  52  Luisen  Strasse,  and 
Carl  Zeiss,  29  Dorotheen  Strasse,  Berlin,  for  the  loan  of  the  blocks  used 
in  illustrating  the  various  Bacteriological  Appliances. 

The  Photomicrographs  and  Photographs  in  this  work  are  original, 
being  taken  from  my  own  specimens  and  cultures,  with  the  exception  of 
Figs.  58,  78,  and  Plate  III,  18  ;  Plate  IV,  20,  24. 


THOS.  BOWHILL. 


BACTERIOLOGICAL  LABORATORY, 
29  CHAMBERS  STREET, 

EDINBURGH,  October  1898. 


ERRATA. 

Page     6,  line  25,  for  "  a  definite,"  read  "  an  indefinite." 
,,      21,    ,,    22,  for  "Nielsen,"  read  "Neelsen." 
,,       27,    ,,    30, for  "symtomatic"  read  "symptomatic." 
„       29,    „     14,  for  "Part  III."  read  "Part  VI." 
,,      39,    ,,    31,  for  "penetrates"  read  "penetrate." 
,,      45,    „      5,  for  "  Iodine  "  read  "  Iodide." 
,,      65,    ,,      5,  for  "  Laborious,"  read  "  Liborius." 
„      87,    ,,    15  ;  page  96,  line  13 ;  page  97,  line  9 ;  page  208,  line  8,  for 

"  pyocyaneus  "  read  "  pyocyanus." 
,,      94,    ,,    10,  for  "Gonorrhoea,"  read  "Gonorrhoeas." 
,,     103,    ,,     41, /or  "saffronine  "  read  "safranine." 

,,     110,    ,,    33, /or  "no  growth  in  CO  2,"  read  "in  an  atmosphere  of  CO2." 
„     113,    „    18, for  "Bacillus  (Edematis  Maligni,"  read  "Bacillus  des 

Malignen  CEdems." 

,,     132,    ,,     33,  for  "  Schultz  "  read  "  Schutz." 
,,     143,    ,,    39,  for  "  Morgenrath's  "  read  "  Morgenroth's." 
„     153,    ,,    34,  for  "separated"  read  "situated." 
„     171,    „      6,/or  "Halz"read"Holz." 

,,     174,    ,,      5  ;  page  175,  line  16,  for  "  Communis  "  read  "  Commune." 
,,     175  (Table  (6),  Widal's  reaction),  for  "  in  contact  with  a  genuine  case 

of  typhoid  fever,  read  "in  contact  with  the  serum 

from  a  genuine  case." 
,,     198,  line  19,  for  "  fleas,"  read  "  flies." 
,,     212,    ,,    33,  fo r  "stongly"  read  "  strongly." 
,,     222,    ,,    29,  for  "  germine  "  read  "  genuine." 
,,     244,    ,,    32,  for  "  haematine  "  read  "  hajmatein." 
,,     254,    ,,    33, /or  "ductu,"  read  "ductus." 
,,     274,    ,,      S,for  "Thierartz,"  read  "  Thierartzliche." 


SUMMARY     OF     CONTENTS 

PAGE 

INTRODUCTION,        .......  1 

THE  CLASSIFICATION  AND  MORPHOLOGY  OF  BACTERIA — 

Flagella  or  Motile  Organs,  Dimensions,  Multiplication,  and 

Spore  Formation,  .....          3-7 

METHODS  OF  STERILIZATION — 

Ordinary  Methods,      ......  7-1 1 

Tyndall's  Discontinuous  Method,        .              .              .              .  11 

Sterilization  of  Post-mortem  Instruments,      .              .              .  12 

PART  I. 

PRINCIPLES  OF  BACTERIOLOGICAL  TECHNIQUE — 

The  Working  Table,  .  .  .  .  .13 

General  Methods  of  Bacteriological  Investigation,     .  .  15 

Methods  of  Preparing  and  Staining  Cover-glass  Specimens, 

The  Hanging-drop,  etc.  ....      15-30 

Methods  of  examining  Sections  of  Organs  and  Tissues  for 
the  detection  of  Bacteria ;  Embedding ;  Section 
Cutting ;  and  Staining  Methods,  .  .  .  30-40 

Stains,  Mordants,  and  Special  Reagents,        .  .  .      40-46 

PART  II. 

THE  PREPARATION  OF  NUTRIENT  MEDIA — 

Bouillon  Media,  ......           47 

Potato  „       .              .              .              .              .              .48 

Peptone  »'.'/•              •              •                           •              .50 

Milk  „                     .              .                            .              .51 

Egg  „ 51 

Gelatine  „                     .              .              .              .              .52 

Agar-Agar  „                     .              .              .                                       53 

Blood  Serum  „                     .              .              .              .              .55 

Special  „                     .              .              .              .             .57 


via 


CONTENTS 


METHODS  OF  CULTIVATING  BACTERIA —  PAOK 

Koch's  Original  Plate  Culture  Method,         .              .              .  58 

Roll  Culture  Methods,           .  6l 

Method  of  Counting  Colonies  in  Roll  Cultures,         .              .  62 

„         obtaining  a  Pure  Culture,             ...  63 

„          Cultivating  Anaerobic  Bacteria,               .              .  64-68 

The  Incubator,  ...  .68 

Special  Reactions  produced  by  Bacteria  during  their  growth,  68-72 

Bacteria  Filter           ......  73 

Inoculation  of  Animals,         .....  74-78 

Post-mortem  Examination  of  Animals,          .              .              .  78 

Methods  of  Examining  Air,  Water,  and  Earth,        .              .  80-85 

Points  to  be  observed  in  describing  an  Organism,  .              .  85 


PART  III. 
SPECIAL  BACTERIOLOGY — 

Bacteria  found  in  Inflammation  and  Suppuration,    .  .      87-102 

The  Streptothrices,  .  .  .  .  .  .102-109 

Anthrax,  Malignant  GEdema,  Symptomatic  Anthrax,  and 

Tetanus,  .  .  .  .  .  .10.9-122 

Bacteria  associated  with  Meat  Poisoning,     .  .  .    122-127 

The  Proteus  Group  of  Bacteria,        ....    127-129 

Glanders,  Bacillus  Orchiticus,  and  African  Farcy,  .  .    129-136 

Tuberculosis,  -.  .  .  .  .  .    136-14$ 

Pseudo-tuberculosis,  .  .  .  .  .149 

Leprosy  and  Smegma  Bacillus,         .  .  .  .152 

Asiatic  Cholera  and  other  Spirilla,   ....    153-162 

Diphtheria  of  Man  and  Animals,      ....    162-169 

Typhoid  Fever,         .  .  .  .  .  .169-174 

Colon  Bacillus,  .  .  .  .  .  .174 

Influenza  and  Pseudo-influenza,         ....    176-179 

The  Septicaemia  Haemorrhagica  Group  of  Bacteria,  .    179-201 

Pleuro-pneumonia  Contagiosa  Bovis,  .  .  .  201 

Foot  and  Mouth  Disease,      .....  203 

Canine  Distemper,    .  .  .  .  .  .204 

Bacteria  found  in  the  Mouth,  ,  .  .  .  205 

„  „  Urine,  .  .  .  205 

Chief  Bacteria  occurring  in  Air,  Soil,  and  Water,    .  .    206-213 

Bacteria  found  in  Milk,         .....    213-220 

„        causing  Acetic  Acid  Fermentation,  .  .  220 

„  „        Butyric         „  .  .221 

„  „        Specific  Changes  in  Beer,  Wine,  and  Sugar,  222-225 

The  Phosphorescent  Bacteria,  ....    225-226 

Thermophilic  and  Drumstick  Bacteria,         .  .  .    226-227 


CONTENTS  ix 
SPECIAL  BACTERIOLOGY — continued  : 

PAGE 

Bacillus   Capsulatus,  Bacillus  Megaterium,  and  Bacterium 

Zopfii,     .......  227-229 

The  Cladothrices,      ......  229-230 

Bacteria  found  in  Leguminous  Nodules,       .              .              .  230-232 

The  Nitrifying  Bacteria,       .              .              .              .              .  232 

The  Nitre-Bacteria,  ......  233-234 


PART  IV. 
THE  HYPHOMYCETES,  OR  MOULD  FUNGI — 

Achorion  Schonleinii,             .....  235 

Tinea  Galli,  .......  236 

Tricophyton  Tonsurans,        .....  236 

Thrush,          .  .  .  .  .  .  .237 

Oidium  Lactis,           .              .              .              .              .              .  238 

Penicillium  Glaucum,             .              .              .              .              .  238 

The  Aspergilli,          ......  239 

Microsporon,              ......  240 

The  Mucors,               .              .              .              .              .              .  241 

Fusisporium  Moschatum,      .              .              .              .  242 


PART  V. 

THE  BLASTOMYCETES,  OR  YEAST  FUNGI — 

Saccharomyces  Cerevisiae,  I.,              .              .              .              .  244 

Ellipsoideus,  I.,  II., ....  245 

Pastorianus,  I.,  II.,  III.,        ...  247 

„  Apiculatus,   .  .  .  .  .248 

„               Anomalus,    .....  248 

„               Marxiaiius,   .              .              .              .              .  248 

„               Membranaefaciens,   ....  249 

„               Exiguus,        .....  249 

„              Acidi  Lactici,            ....  249 

Mycoderma  Cerevisiae  et  Vini,           ....  249 

The  Torulae, .......  250 

PATHOGENIC  BLASTOMYCETES — 

Saccharomyces  Hominis,       .....  250 

„               Lithogenes,                .              .              .              .  251 
„               Neoformans,               .              .              .              .251 

„               Subcutaneus  Tumefaciens,  .  251 

„              in  Hydrophobia,       ....  252 


PAOK 


x  CONTENTS 

PART  VI. 
THE  PROTOZOA,  OR  ANIMAL  PARASITES — 

Amoeba  Coli,                 .              .             ..  -                                       253 

Paramaecium  Coli,        .  254 

Coccidia,           .              .  254 

Coccidium  Oviforme,  .  254 

Plasmodium  Malariae,  255 

Southern  or  Texas  Cattle  Fever,.  261 

Australian  Tick  Fever,  263 

Ixodic  Anaemia  in  Jamaica,     .              .  263 
Babesia  Bovis,              ......         264 

„       Ovis,  .  265 

Bovine  Malaria,            .  265 

Klossia  Soror,               .              .  265 

PHOTOMICROGRAPHY,             .  .                       265 

APPENDIX,                ......  271 


LIST    OF    ILLUSTRATIONS 


FIG. 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 
10. 
11. 
12. 
13. 
14. 
15. 
16. 
IT. 
18. 
19. 
20. 
21. 


Hot-Air  Sterilizer,     ... 

Koch's  Steam  Sterilizer,       ..... 

BowhilFs          ,,  . 

Autoclave,     . 

Nuttall's  Microscopic  Thermostat, 

Altman's  Hand  Centrifuge, 

Jung's  Students'  Microtome,  .... 

Schauze's  Microtome,  ..... 

Koch's  Apparatus  for  Congealing  Blood  Serum,   . 

„      Plate  Culture  Apparatus,     . 

Esmarch's  Apparatus  for  Counting  Roll  Culture  Colonies, 
Wolffhiigel's  Counting  Apparatus, 
Liborious's  Tube  for  Anaerobic  Cultures,  . 
Kitasato's  Bottle  „  „          .  .  . 

Botkin's  Apparatus  for  Anaerobic  Plate  Cultures, 
Kipp's  Hydrogen  Apparatus, 
Buchner's  Tube  for  Anaerobic  Cultures,     . 
Incubator,      ....... 

Smith's  Fermentation  Tube, 
Dunbar's         ,,  ,,  . 

22.  Reichel's  Bacteria  Filter,      ..... 

23.  „  

24.  Hesse's  Apparatus  for  examining  Air, 

25.  Air  Pump  for  use  in  Petri's  Method, 

26.  Fraenkel's  Earth  Borer,        ..... 

27.  Streptococcus  of  Erysipelas, 

28.  Diplococcus  Pneumonias,     . 

29.  Bacillus  Pneumonias, 

30.  Micrococcus  Tetragenus, 

31.  Actinomyces  Bovis— culture, 

32.  ,  in  section,       .... 

33.  ,  culture, 

34.  Baci  lus  Anthracis,  *  Leptothrix,'    . 

35.  and  capsules,  . 

36.  stab  culture,    . 

37.  involution  forms, 

38.  QEdematis— stab  culture, 
39. 

40.  Anthracis  Symptomatici — stab  culture,    . 

41.  ,,  ,,  and  Spores, 

42.  Tetani— stab  culture, 
43  ,,        and  Spores, 

44.  ,,        from  point  of  inoculation, 

45.  Proteus  Vulgar  is— swarming  islands, 

46.  Mallei  in  section  of  nodule, 


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LIST  OF  ILLUSTRATIONS 


FIG. 

47. 

48. 

49. 

50. 

51. 

52. 

53. 

54. 

55. 

56. 

57. 

58. 

59. 

60. 

61. 

62. 

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105. 


Bacillus  Tuberculosis  in  sputum,     . 

glycerine  agar  culture, 
in  culture,       .... 
lung  of  a  cow, 

from  horse,    .... 
spleen  of  a  pig, 
Scrofulous  Gland  from  neck  of  a  pig, 
Tubercular  Spleen  from  a  pig, 

Bacillus  of  Avian  Tuberculosis,        .... 
,,  Cholerae  Asiaticae,          .... 

Vibrio  Rugula  with  Flagella, 
Spirillum  Undula        ,, 
Bacillus  Diphtherias — culture, 
Chickens  affected  with  Avian  Diphtheria, 
Bacillus  Typhi  Abdominalis— stab  culture, 
,,  ,,  ,,  from  culture, 

Bacillus  Coli  Commuins— stab  culture, 

,,  ,,  from  culture,      . 

of  Influenza  in  Sputum,  .... 
of  Fowl  Cholera,  ..... 
Phasiani  Septicus,  .... 

of  Swine  Fever — spleen  of  a  pig,  . 
,,  culture,  . 

Stomach  of  Pig  showing  Swine  Fever  Ulcers, 
Broncho-Pneumonia  Suis— lung  of  pig, 
Bacillus  of  Swine  Fever — stab  culture, 
Schweine-seuche 

,,  Swine  Erysipelas  ,, 

,,          Mouse  Septicaemia        ,, 

,,          Bubonic  Plague — agar  culture, 

,,  ,,  spleen  of  mouse, 

„          Broncho-pneumonia  Bo  vis, 

,,      Subtilis  and  Spores,  .... 

„  with  Flagella.  .... 
Vibrio  Metschnikoff  in  pigeon's  blood, 
Trommelschlager  Bacilli,  ..... 
Bacillus  Megaterium  and  Spores,  .... 
Cladothrix  found  in  water,  ..... 
Achorion  Schonleinii — agar  culture, 

,,  ,,  section  of  agar  culture, 

Tricophyton  Tonsurans,      ,,  ,, 

Oidium  Lactis,         ...... 

Penicillium  Glaucum,  ..... 

Aspergillus  Niger,    ...... 

,,  Fumigatus,        ..... 

Mucor  Corymbifer,  ...... 

,,      Mucedo,        ...... 

Fusisporium  Moschatum,     ..... 

Saccharomyces  Cerevisiae  I.  and  Ascospores, 
Coccidium  Oviforme,  stained,         .... 

,,  ,,          unstained,     .... 

Pyrosoma  Bigeminum  in  liver  of  ox 

Klossia  in  a  Snail's  Liver,   ..... 
Zeiss's  large  Photomicrographic  Apparatus, 

,,       special  Microscope  and  Stand  for  Photomicrography, 
Special  Centering  Achromatic  Condenser. . 
Projection  Eye-piece,  ..... 

Plates  I,  II,  III,  and  IV. 


PAGE 

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At  end. 


INTRODUCTION. 

THE  micro-organisms  causing  putrefaction,  fermentation,  and  in- 
fectious diseases  belong  partly  to  the  lower  vegetable,  and  partly  to 
the  lower  animal  kingdom,  and  are  divided  into  four  groups  : — 

1.  Hyphomycetes^  or  Mould  Fungi. 

2.  Blastomycetes,  or  Yeast  Fungi. 

3.  Schizomycetes,  or  Bacteria. 

4.  The  Protozoa. 

1.  The  Hyphomycetes  consist  of  cells  multiplying  only  by  growth 
of  the  distal  or  point  cells,  and  in  this  manner  forming  threads  or 
hyphae,  i.e.,  chains  of  cells.     The  fully-developed  mould  consists  of  a 
mycelium  (which  might  be  compared  to  the  roots  of  higher  plants), 
and  of  fruit  hyphse  which  develop  out  of  the  former,  and  bear  on 
their  extremities  the  spores  or  conidia.     The  moulds  in  the  absence  of 
oxygen  frequently  form    cloudy  mycelial    masses  within  the  culture 
media.     Spores  are  only  formed  on  the  surface  of  media  in  contact 
with  oxygen.     The  Hyphomycetes  possess  no  chlorophyll. 

2.  The  Blastomycetes  consist  of  ovoid  or  round  cells,  multiplying  by 
bud-like   processes   from    the  mother  cells,  also  at    times  by  spore 
formation.     There  is  reason  to  believe  they  are  allied  to  the  mould 
fungi.     If  the  newly-formed  cells  are  not  detached,  a  conglomerate 
cell  of  buds  is  formed.     On  unfavourable,  strongly  alkaline,  and  sugar- 
free  media  a  mycelium  is  developed. 

3.  The  Schizomycetes  or  Bacteria  are  a  large  group  of  unicellular 
organisms  developing  from  pre-existing  cells  of  the  same  species  ;  they 
never  appear  spontaneously,  and  multiply  by  a  process  of  transverse 
division. 

They  are  spherical,  oval,  rod-like,  and  spiral  in  shape,  and  are 
generally  devoid  of  chlorophyll  or  green  colouring  matter  possessed 
by  the  higher  plants ;  owing  to  this  circumstance  they  are  forced  to 
obtain  their  nutritive  materials  from  organic  matters,  and  therefore 
exist  in  either  saprophytic  or  parasitic  conditions. 

A 


2  PRACTICAL  BACTERIOLOGY 

A  saprophyte  may  be  defined  as  an  organism  obtaining  its  nutri- 
tion from  dead  organic  matter,  whilst  a  parasite  exists  at  the  expense 
of  some  other  living  organic  creature,  known  as  its  host,  and  strictly 
speaking  cannot  develop  upon  dead  matter. 

There  is,  however,  a  group  of  so-called  'facultative''  saprophytes 
and  parasites  which  possess  the  power  of  accommodating  themselves 
to  existing  surroundings,  at  one  time  leading  a  parasitic,  and  at 
another  time  a  saprophytic  form  of  existence. 

Decomposition,  putrefaction,  and  fermentation  result  from  the 
activities  of  saprophytic  bacteria,  whilst  the  parasitic  bacteria  cause 
changes  in  the  tissues,  resulting  in  disease  processes,  or  causing  the 
death  of  their  host.  It  has  been  found  convenient  in  classifying 
bacteria  to  describe  their  chief  characteristics  by  the  following 
terms : — 

Chromogenic,  for  pigment-producing  bacteria. 
Photogenic,  for  phosphorescent  or  light-producing  bacteria. 
Zymogenic,  for  bacteria  concerned  in  the  various  fermentations. 
Saprogenic,  for  bacteria  producing  putrefaction. 
Pyogenic,  for  bacteria  producing  suppuration. 
Thiogenic,  for  those  converting  sulphuretted  hydrogen  into  higher 
sulphur  compounds. 

There  is  another  very  important  saprophytic  group  which  com- 
prises the  so-called  '  nitrifying '  and  '  denitrifying '  bacteria.  The 
4  nitrifying '  group  oxidizing  ammonia  to  nitrous  and  nitric  acids  ;  the 
4  denitrifying '  group  reducing  nitric  acid  to  nitrous  acid  and  ammonia. 
Through  their  association  (symbiosis)  with  the  nitrifying  bacteria  and 
the  activity  of  the  latter,  as  far  as  we  know,  leguminous  plants  are 
enabled  to  make  up  their  nitrogen  deficit  in  part  from  the  free 
nitrogen  of  the  air. 

This  important  discovery  gave  to  free  atmospheric  nitrogen  a 
biological  significance  heretofore  denied. 

Owing  to  the  absence  of  chlorophyll,  bacteria  must  have  the  carbon 
and  nitrogen  necessary  for  their  growth  in  the  form  of  decomposable 
organic  substances,  and  the  most  favourable  media  for  their  develop- 
ment are  neutral  or  very  slightly  alkaline  solutions  of  proteid 
materials  in  one  form  or  another. 

Bacteria  growing,  multiplying,  and  performing  definite  functions  in 
the  absence  of  oxygen,  and  to  the  existence  of  which  oxygen  is 
positively  harmful,  are  known  as  'Anaerobic1  bacteria,  in  contra- 
distinction to  the  Aerobic  group,  for  the  proper  performance  of  whose 
functions  free  oxygen  is  essential. 


INTRODUCTION  3 

Many  organisms  can  accommodate  themselves  equally  well  to  both 
these  modes  of  existence,  —exposed  to  the  air  they  are  '  aerobic,"1  in  its 
absence  they  become  '  anaerobic.''  Bacteria  possessing  this  faculty  are 
known  as  '  aero-anaerobic '  or  'facultative  "*  organisms. 

The  most  favourable  temperature  for  the  development  of  bacteria 
pathogenic  for  warm-blooded  animals  is  that  of  the  human  body,  viz., 
37*5°  C.,  while  the  so-called  normal  water  bacteria  grow  best  at  about 
20°  C.,  and  the  '  thermophilic '  bacteria  develop,  according  to  some 
authorities,  as  low  as  34°  to  44°  C.,  and  as  high  as  70°  to  75°  C. 
From  this  it  will  be  seen  that  the  range  of  the  activity  of  bacterial 
growth  depends  upon  specific  differences  and  may  have  wide  limits : 
some  growing  best  at  low,  others  at  high,  and  others  at  medium  tem- 
peratures. Corresponding  to  this  we  find  that  the  limits  are  defined  by 
minimum,  optimum,  and  maximum  temperatures  for  growth.  The 
minimum,  when  growth  is  just  possible ;  optimum,  when  the  growth  is 
most  luxurious ;  and  maximum,  the  highest  temperature  at  which  the 
organism  will  develop. 

Besides  a  suitable  temperature,  bacteria  require  for  their  develop- 
ment moisture  and  a  medium  of  suitable  composition  and  reaction. 

Bacteria  are  also  influenced  in  a  varying  degree  by  light — most  forms 
develop  by  far  the  best  in  the  dark.  It  is  therefore  important  to 
have  a  dark  closet  in  the  laboratory  for  the  storing  of  cultures,  and  it 
is,  moreover,  known  that  cultures  retain  their  vitality  and  virulence 
longer  when  maintained  at  low  temperatures. 

4.  The  Protozoa  are  one-celled  animal  organisms,  usually  consider- 
ably larger  than  the  largest  bacteria,  on  which  they  frequently  feed. 
The  cell  body  is  differentiated  as  a  homogeneous  ectoplasm  and  a 
granular  entoplasm  containing  vacuoles,  also  one  or  more  nuclei. 
Some  protozoa  possess  motile  organs  or  flagella,  others  possess 
pseudopodia,  others  cilia.  They  develop  by  fission  or  by  spore 
formation.  They  may  coalesce  and  form  so-called  plasmodia,  and  as 
in  the  bacteria  the  spores  may  be  more  resistant  than  the  active 
organism. 


THE  CLASSIFICATION  AND  MORPHOLOGY 
OF  BACTERIA. 

The  following  simple  classification  of  bacteria  has  been  found  con- 
venient by  medical  bacteriologists,  though  perhaps  not  quite  correct 


4  PRACTICAL  BACTER  OLOGY 

from  a  botanical  point  of  view.     The  three  principal  divisions  are  as 
follows : — 

1.  Cocci  or  Micrococci,  or  round  organisms. 

%.  Bacilli,  or  rod  organisms. 

3.  Spirilla,  or  spiral  organisms. 

1.  The  Cocci  or  Micrococci  are  subdivided  as  follows  : — 

(1.)  Staphylococci — growing  like  bunches  of  grapes. 

(2.)  Streptococci — growing  in  chains. 

(3.)  Diplococci — growing  in  pairs. 

(4.)  Tetrads — developing  in  fours. 

(5.)  Sarcinae — dividing  into  fours,  eights,  etc.,  as  cubes. 

All  spherical  forms  belong  to  this  group,  the  isolated  individual 
member^  of  which  are  practically  of  the  same  diameter  in  all  directions. 

2.  The  Bacilli  include  all  straight,  rod-like  bacteria  in  which  one 
diameter  is  always  greater  than  the  other.     Many  of  the  organisms 
belonging  to  this  group  in  the  course  of  development  deviate  from 
the  simple  rod  shape,  and  when  very  short,  i.e.,  multiplying  rapidly, 
appear  like  micrococci ;  the  ends  of  rods  also  vary  in  shape  according 
to  the  particular  organism,  being  either  blunt  or  rounded. 

3.  The  Spirilla  are  subdivided  as  follows  : — 

(1.)  Vibrio,  or  short  spirals. 

(2.)  Spirillum,  or  long  rigid  spirals. 

(3.)  Spirochaete,  or  long  flexible  spirals. 

Different  bacteria  may,  moreover,  be  characterised  by  the  following 
peculiarities: — They  may  form  Zooglcea,  by  which  we  understand 
bacterial  aglomerations  of  bacteria  in  large  numbers  enclosed  in  an 
amorphous  matrix,  which  may  give  great  tenacity  to  the  bacterial 
mass. 

By  Ascococcus  we  understand  cocci,  associated  in  large  numbers  in 
an  amorphous  matrix,  and  enclosed  in  an  enveloping  membrane. 

Some  bacteria  possess  a  distinct  capsule,  and  are  known  as  Capsule 
cocci  and  Capsule  bacilli,  the  capsule  being  more  or  less  evident, 
depending  upon  the  conditions  of  growth. 

By  Leptothrix  we  understand  long  undulating  rods. 

By  Cladothrix,  long,  straight  and  branching  rods. 

By  Streptothrix,  organisms  that  in  their  structure  resemble  at  one 
time  the  thread  fungi,  and  at  other  times  the  bacteria;  like  the  mould 
fungi,  they  form  cylindrical  threads  out  of  round  cells,  which  branch 
dichotomously,  finally  becoming  visible  to  the  naked  eye  as  irregular 
radiating  thread  masses  or  mycelia. 

Involution  Jorms  of  bacteria  usually  occur  under  conditions  un- 


CLASSIFICATION  AND  MORPHOLOGY  5 

favourable  to  the  nutrition  of  the  organism  causing  degeneration, 
whereby  it  acquires  abnormal  aspects,  as  in  old  cultures  or  in  media  of 
unsuitable  composition. 

Pleomorphism. — Recent  researches  show  that  the  same  organism 
may  assume  very  different  aspects,  and  appear  either  as  bacilli  or 
micrococci.  However,  pleomorphic  organisms  are  the  exception,  and 
it  is  very  unsafe  for  the  beginner  to  assume,  when  he  finds  organisms 
in  his  cultures  presenting  different  appearances,  that  he  is  dealing 
with  pleomorphic  bacteria. 

FLAGELLA   OR   MOTILE   ORGANS. 

All  motile  bacteria  are  provided  with  flagella,  which  are  distin- 
guished according  to  their  number  and  position,  as  follows : — 

1.  Monotricha,  one  flagellum  at  one  pole. 

2.  Amphitricha,  one  flagellum  at  each  pole. 

3.  Lophotricha,  a  bundle  of  flagella  at  one  pole. 

4.  Peritricha,  many  flagella  given  off  around  the  periphery  of  the 

organism. 

The  above  terminology  being  borrowed  from  that  applied  to  the 
Protozoa. 

(For  special  method  of  demonstrating  Flagella,  see  §§  23,  24.) 

THE   DIMENSIONS   OF   BACTERIA. 

These  vary  as  much  as  their  form,  and  are  expressed  in  microns. 
A  micron  is  the  one- thousandth  part  of  a  millimetre,  and  is  desig- 
nated by  the  Greek  letter  /x, — i.e..  Bacillus  Anthracis,  3-10  /x  long,  and 
1-1 '2  fj.  broad.  Bacillus  Tuberculosis,  l'5-4  /x  long,  and  only  0*4  /x 
broad,  etc. 

MULTIPLICATION. 

1.  Micrococci  develop  by  simple  fission  (dividing  into  two  trans- 
versely). 

2.  Staphylococci,  when  forming  characteristic  grape-like  clusters  by 
growth  and  division  in  different  directions. 

3.  Streptococci  develop  in  one  direction  only,  producing  chains  of 
varying  length. 

4.  Sarcince  divide  regularly  in  three  directions  of  space,  but  instead 
of  separating  as  single  cells,  remain  together  in  masses  like  bales  of 
cotton,  the  individual  members,  pairs,  cubes,  etc.,  being   each   en- 
veloped in  a  capsule. 


6  PRACTICAL  BACTERIOLOGY 

5.  The  Bacilli  multiply  by  a  process  of  division,  which  is  transverse 
to  their  direction  of  growth.  The  mother  cell,  having  become  elon- 
gated, develops  into  two  daughter  cells — in  this  way  chains  of  indi- 
viduals are  formed.  The  length  of  these  chains  vary  in  the  different 
species,  and  is  also  influenced  by  different  conditions.  Long  chains  of 
individuals,  when  this  division  is  not  very  distinct,  may  present  a 
homogenous,  thread-like  appearance  in  unstained  specimens.  The 
rapidity  of  the  growth  is  naturally  variable,  according  to  the  species, 
nature  of  the  medium,  temperature,  etc.  The  most  rapid  growth 
observed  has  been  the  division  of  a  mother-cell  into  two  daughter- 
cells  within  fifteen  to  twenty  minutes. 

SPORE   FORMATION. 

Some  bacteria  develop  what  are  known  as  spores;  by  spores  we 
understand  a  resting  stage  in  the  life-history  of  an  organism.  Spores 
are  either  formed  within  the  mother-cells  (endogenous),  or  certain 
individual  cells  prove  more  resistant.  Endogenous  spores  are  formed 
in  the  body  of  the  bacilli,  usually  under  conditions  unfavourable  to 
their  growth, — that  is,  when  they  have  exhausted  their  food  supply  : 
this  is,  however,  not  alwrays  the  case.  Spores  are  usually  resistant,  at 
times  highly  so,  to  influences  which  destroy  vegetative  forms.  They 
withstand  drying,  at  times  for  many  years,  and  great  elevations  of 
temperature,  as  well  as  the  effect  of  chemical  agents.  Brought  under 
favourable  conditions  the  spores  germinate  again  to  the  vegetative 
form  or  bacillus  ;  they  do  not  as  such  multiply  of  themselves,  but  give 
rise  each  to  a  single  bacillus  which,  dividing,  gives  rise  to  a  definite 
number  of  individuals. 

The  changes  in  bacilli  about  to  form  spores  are  as  follows : — 

1 .  The  protoplasm  loses  its  normal  homogenous  appearance,  and 

granular  refractive  points  of  irregular  shape  and  size 
appear. 

2.  The   above   granular    refractive   points    finally    coalesce,    the 

remainder  of  the  cell  remaining  clear  and  transparent. 
Free  granules  may  remain  in  the  vicinity  of  the  spore,  as 
if  not  required  for  its  development. 

3.  The  complete  spore  appears  as  an  oval,  highly  refractive,  glis- 

tening body,  easily  differentiated  from  the  remainder  of 
the  cell,  which  now  consists  only  of  cell  membrane  and 
detritus. 

4.  The    cell    membrane    eventually  disappears,    liberating    the 

spore. 


CLASSIFICATION  AND  MORPHOLOGY  7 

A  single  cell  usually  produces  but  one  spore,  which  may  be  located 
in  the  centre  or  at  the  extremity  of  the  mother-cell.  The  cell  which 
produces  a  spore  may  assume  the  shape  of  a  drum-stick  or  a  spindle 
(known  as  a  Clostridium).  Spores  cannot  be  stained  by  the  ordinary 
methods  employed  for  bacteria  (for  special  methods  of  staining  Spores 
see  §§  26,  27). 

A  spore  about  to  develop  into  a  bacillus  exhibits  the  following 
phenomena : — 

1.  It  gradually    loses  its  highly  refractive  appearance,  enlarges, 

and  appears  to  assume  a  consistency  approaching  to  that 
of  the  bacillus. 

2.  The  membrane  of  the   spore  is  ruptured,  and    the  bacillus 

grows  out  of  the  aperture. 

3.  In  some  organisms  the  separation  of  the  spore  capsule  is  more 

evident  than  in  others,  and  in  this  case  the  remains  of  the 
capsule  may  adhere  for  some  time  to  the  young  bacillus. 
It  is  on  account  of  the  resistance  of  the  spores  that  the 
elaborate  means  of  sterilization  about  to  be  described  are 
necessary  for  obtaining  media  free  from  germs,  by  which 
in  turn  we  can  obtain  pure  cultures — that  is,  separate  culti- 
vations of  single  species  of  bacteria.  The  resistance  of 
the  spores  is  due  to  a  very  dense  and  impenetrable  mem- 
brane, as  well  as  to  the  fact  that  the  protoplasm  contains 
less  water  than  that  of  the  vegetative  form.  More  water 
in  conjunction  with  protoplasm  lowers  the  temperature  at 
which  it  coagulates. 


METHODS    OF    STERILIZATION. 
STERILIZATION  BY  HEAT. 

Sterilization  may  be  accomplished  by  subjecting  the  articles  to  be 
treated  to  high  temperatures,  either  in  a  moist  or  dry  state.  Success- 
ful sterilization  by  dry  heat  cannot  usually  be  accomplished  at  a 
temperature  lower  than  150°  C.,  and  the  objects  should  be  subjected 
to  this  temperature  for  not  less  than  one  hour.  The  apparatus  used 
for  hot-air  sterilization  is  double-jacketed,  and  made  of  strong  sheet- 
steel,  preferably  with  a  copper  bottom,  and  having  two  perforated 
movable  shelves.  On  the  top  there  is  a  regulating  slide,  by  which  a 
current  of  air  through  the  apparatus  may  be  secured,  and  two  tubes, 


8  PRACTICAL  BACTERIOLOGY 

one  for  a  thermometer  and  the  other  for  a  thermo-regulator — which 
is  usually  not  necessary — and  two  eyelits  for  suspending  it  to  the  wall, 
or  it  can  be  placed  on  a  stand  (see  Fig.  1).  When  suspended  against 
the  wall  a  sheet  of  asbestos  is  necessary  to  protect  the  wall.  It  is  also 
important  that  the  copper  bottom  of  the  apparatus  should  be  detach- 
able, thus  enabling  a  new  one  to  be  fitted  without  difficulty. 

In  bacteriological  work  sterilization  by  dry  heat  is  limited  to  such 
articles  as  glass  flasks,  plates,  small  dishes,  test-tubes,  pipettes,  cotton 
wadding,  and  such  metal  instruments  as  are  not  seriously  injured  by 
the  high  temperature.  The  above  articles  are  sterile  when  heated  to 
150°  C.  for  one  hour.  The  usual  method  is  to  remove  the  Bunsen 
burner  when  the  thermometer  registers  170°  C.,  and  allow  the 
apparatus  to  cool  before  opening,  otherwise  glass  ware  may  fly  in 
pieces. 


FIG.  1.— Hot- Air  Sterilizer. 


FIG.  2.— Koch's  Steam  Sterilizer. 


Fluids,  culture  media,  potatoes,  etc.,  in  fact,  anything  that  would 
be  destroyed  by  the  great  heat  of  the  hot-air  chamber,  are  sterilized 
in  the  '  Koch  Steam  Sterilizer'1  (see  Fig.  2),  or  the  '  Arnold  Steam 
Sterilizer.'' 

The  Arnold  apparatus  possesses  advantages  not  obtainable  with 
the  Koch  apparatus.  It  is  smaller  but  quicker,  and  is  more  suitable 


METHODS  OF  STERILIZATION  9 

for  rapid  work,  the  Koch  apparatus  requiring  a  long  time  to  obtain 
the  temperature  of  100°  C.,  from  which  moment  the  period  of  steriliza- 
tion can  be  estimated. 

Messrs  Lautenschlager,  Berlin,  have  recently,  at  the  writer's  sug- 
gestion, constructed  a  useful  portable  steam  sterilizer,  costing  27/- 
(see  Fig.  3),  showing  the  apparatus  with  the  outer  cover  removed,  and 
the  inner  lid  of  the  steam  chamber  slightly  raised.  The  outer  cover 
can  also  be  used  without  the  inner  lid  when  a  temperature  lower  than 
100°  C.  is  required ;  a  place  for  a  thermometer  being  provided. 

Fig.  4  shows  a  section  of  the  apparatus,  P  being  the  water 
reservoir,  and  the  boiling  chamber  being  constructed  similar  to  the 
Arnold  apparatus.  A  and  D  show  the  junction  of  the  steam  and 
water  chambers  by  means  of  a  strong  detachable  telescopic  joint, 
enabling  the  water  chamber  to  be  cleansed,  and  also  facilitating  the 
packing  of  the  apparatus  during  transportation.  The  size  of  the 
opening  admitting  the  steam  into  the  steam  chamber  is  regulated 
with  a  diaphragm  fitted  on  the  inside  of  the  steam  chamber. 


FIG.  3. 


FIG.  4. 


Bowhill's  Steam  Sterilizer. 


Whereas  the  above  means  of  sterilization  at  100°  C.  suffice  for 
ordinary  purposes,  an  apparatus  for  sterilization  by  steam  under 
pressure,  known  as  an  Autoclave  (see  Fig.  5),  is  at  times  very  useful, 


10 


PRACTICAL  BACTERIOLOGY 


for  the  reason  that  the  whole  process  of  sterilization  may  be  short- 
ened from  several  hours  to  as  many  minutes.     It  can  only  be  used, 


Fiu.  5.— Autoclave. 


however,  for  media  which  remain  uninjured  by  the  exposure  to  the 
higher  temperature  obtained  in  the  autoclave. 


METHODS  OF  STERILIZATION  11 


SIMPLE  STEAM  STERILIZER  'NOVY/ 

The  lower  part  of  the  apparatus  consists  of  an  ordinary  water 
bath,  7  to  8  inches  in  diameter ;  the  upper  part  of  a  copper  pail 
8  inches  high  and  8  inches  in  diameter,  with  a  perforated  bottom, 
and  lid  with  a  tube ;  copper  rings  are  soldered  in  the  interior  of  the 
pail  to  prevent  the  tube  touching  the  sides,  otherwise  the  cotton 
plugs  would  become  saturated  with  the  condensed  steam.  The  pail  is 
filled  with  flasks  or  tubes,  and  placed  over  the  water  bath,  in  which 
the  water  should  be  boiling.  In  five  to  seven  minutes  steam  will 
actively  issue  from  the  tube  in  the  cover,  showing  that  the  interior 
temperature  has  reached  100°  C.  With  the  apparatus  a  student  can 
attend  to  any  needed  steam  sterilization  at  his  own  table,  and  thus  save 
much  time.  The  general  usefulness  of  the  apparatus,  its  compactness, 
cheapness,  and  the  saving  in  the  gas,  will  recommend  it  to  those 
practitioners  who  desire  to  equip  a  small  laboratory. 

A  small  steam  sterilizer  may  be  readily  improvised  by  standing  an 
inverted  funnel  of  appropriate  size  upon  the  water  bath.  If  the 
funnel  is  of  glass  it  is  well  to  surround  it  writh  a  towel,  to  prevent  its 
cooling  too  rapidly  on  removal.  Dr  Nuttall  states  that  he  has  used 
this  simple  apparatus  frequently  during  the  last  ten  years  and  found 
it  very  useful. 

TYNDALUS    DISCONTINUOUS    METHOD    OF 
STERILIZATION. 

We  have  stated  above  that  certain  bacteria  form  spores  which  are 
highly  resistant,  whereas  the  vegetative  form  of  the  organism  is  more 
sensitive  to  heat,  chemical  agents,  etc.  TyndalPs  method  of  discon- 
tinuous sterilization  depends  upon  the  principle  that  bacteria  develop- 
ing from  spores  in  the  nutrient  media  are  destroyed  by  a  relatively 
short  exposure  to  a  temperature  of  100°  C.,  at  which  temperature 
the  spores  are  not  affected.  To  overcome  this  difficulty  presented  by 
the  resistance  of  the  spores,  the  tubes  of  media  or  other  materials  are 
placed  in  a  steam  sterilizer  at  100°  C.  for  thirty  minutes.  The  first 
heating  not  having  killed  the  spores,  the  material  is  placed  at  ordinary 
room  temperature,  or  better,  at  37°  C.,  when  any  living  spores  quickly 
vegetate.  The  bacteria  to  which  the  spores  give  birth  are  killed  by  a 
second  heating,  for  twenty  or  thirty  minutes,  on  the  following  day. 
The  above  operation  repeated  three,  four,  or  five  times,  ends  in  the 
certain  sterilization  of  the  media,  all  the  spores  having  developed  into 
bacilli,  and  as  such  destroyed. 


12  PRACTICAL  BACTERIOLOGY 

The  process  of  fractional  sterilization  at  low  temperatures  is  con- 
ducted in  a  somewhat  similar  manner  to  TyndalPs  method,  but  re- 
quires a  greater  number  of  exposures  to  a  temperature  not  exceeding 
68°  to  70°  C.,  and  is  employed  to  sterilize  easily  decomposable 
materials  which  would  be  rendered  unfit  for  culture  purposes  by 
steam — such  as  blood  serum.  Sterilization  by  steam  can  also  be 
practised  by  what  is  known  as  the  '  direct  method,1  by  a  single  ex- 
posure in  the  Koch  Steam  Sterilizer,  but  a  prolonged  exposure  is 
necessary — at  times  several  hours — to  obtain  complete  sterilization. 

When  an  autoclave  is  used,  a  single  exposure  of  fifteen  minutes  is 
sufficient  to  practically  destroy  all  bacilli  and  their  spores,  provided 
the  thermometer  registers  122°  C.  The  autoclave  when  used  must 
either  remain  closed  until  cool,  or  until  the  gauge  indicates  that 
pressure  no  longer  exists,  for  if  opened  when  the  steam  within  is  still 
under  pressure,  the  steam  will  escape  so  rapidly  that  all  fluids  within 
the  chamber  thus  suddenly  relieved  of  pressure,  will  boil  violently,  and 
as  a  rule  boil  quite  out,  of  the  tubes,  blowing  out  the  plugs. 

Open  vessels  before  sterilization  are  closed  with  cotton-wool  plugs 
or  stoppers.  Glass  plates  and  pipettes  are  sterilized  in  copper  or 
sheet-iron  boxes  specially  manufactured  for  the  purpose,  and  removed 
when  required.  It  is  convenient  to  wrap  each  pipette  in  paper. 

Petri  dishes  are  most  conveniently  sterilized  in  a  special  cylindrical 
copper  box  with  a  capped  lid,  there  being  a  round  hole  in  the  box 
and  in  the  lid.  These  holes  are  placed  opposite  each  other  during 
sterilization,  thus  allowing  a  current  of  hot  air  to  circulate  in  the 
interior  of  the  cylinder.  When  the  process  of  sterilization  is  com- 
pleted, the  lid  is  slightly  turned,  closing  the  holes.  The  interior  of 
the  cylinder  is  fitted  with  a  special  apparatus,  enabling  one  or  any 
number  of  dishes  to  be  withdrawn  when  required. 


STERILIZATION  OF  POST-MORTEM  INSTRUMENTS. 

1.  Wipe  the  dirty  knife  with  a  piece  of  cotton  wool  and  dip  in  a 
strong  solution  of  caustic  soda. 

2.  Wash  in  alcohol,  heat  in  a  flame,  boil  in  strong  soda  solution,  or 
place  in  the  steam  sterilizer. 

Instruments  may  be  quite  conveniently  sterilized  in  the  laboratory 
by  dipping  them  in  benzine  and  inflaming  the  latter.  The  steriliza- 
tion is  thus  completed  without  exposing  the  instruments  to  as  high  a 
temperature  as  when  they  are  drawn  through  the  flame. 


PART    I. 

PRINCIPLES    OF    BACTERIOLOGICAL 
TECHNIQUE. 

THE   WORKING  TABLE    OR  BENCH. 

§  I.  The  bench  or  work-table  must  be  horizontal  and  steady,  4  feet 
by  2  feet  4  inches  on  top,  and  2  feet  4  inches  high.  The  surface  of 
the  table  is  covered  with  a  piece  of  plain  brown  linoleum,  which  can 
be  renewed  when  necessary.  Piano  stools  make  the  best  seats,  being 
adjustable  for  height  and  convenient  for  any  desired  position.  A 
sheet  of  plate  or  other  thick  glass  is  laid  on  the  left  hand  side  of  the 
table,  with  pieces  of  black  and  white  paper  underneath,  forming  a 
background ;  two  pieces  of  coloured  glass  can  also  be  used,  one 
piece  milk-white  and  the  other  black,  according  as  the  specimens 
under  examination  are  stained  or  unstained.  Watch  glasses,  capsules, 
slides,  etc.,  under  naked-eye  examination  are  placed  on  either  the 
white  or  black  surface. 

1.  Unstained  specimens  or  sections  are  best  seen  on  the  black. 

2.  Stained  specimens  on  the  white. 

3.  The   black   surface   is  the   most    suitable    for    isolating    the 

caseous  portions  of  suspected  sputum. 

Opposite  and  within  easy  reach  of  the  operator  a  rack  is  placed 
to  hold  the  stains  and  reagents  in  daily  use  (see  §  2).  A  solid 
block  of  wood  with  a  number  of  circular  holes  bored  in  it  is  the  most 
convenient  rack.  A  Bunsen  burner  is  placed  on  the  right  hand  side 
of  the  table,  but  in  the  absence  of  gas  a  spirit  lamp  answers  the 
purpose.  On  a  shelf  3  or  4  feet  above  the  right  side  of  the  table  a 
large  bottle  of  distilled  water  is  placed,  with  a  rubber  tube  and 
water  pinch-cock  attached  to  its  distal  extremity,  descending  to  a 
circular  glass  dish  within  easy  reach  of  the  operator,  for  washing 
excess  stain,  etc.,  from  specimens. 


14  PRACTICAL  BACTERIOLOGY 

§  II.  The  following  articles  are  also  placed  on  the  table  :— 

1.  A  large  glass  dish,   6  inches  by  7  inches,  with  a  cover,  con- 

taining Disinfecting  Solution  (see  §  66),  for  the  temporary 
disposal  of  old  cultures,  virulent  material,  etc. 

2.  A  small  glass  dish,  3|  inches  by  2  inches,  containing  Cleansing 

Solution  (see  §  65),  for  the  temporary  disposal  of  dirty 
slides  and  cover-glasses. 

3.  A  covered  glass  dish,  containing  vaseline  and  a  small  camePs- 

hair  brush,  for  preparing  hanging-drop  cultures. 

4.  One  large  test-tube  stand. 

5.  One  small  test-tube  stand. 

6.  One  small  filter  stand. 

7.  One  bottle  of  Canada  balsam  dissolved  in  xylol. 

8.  One  small  bottle  of  immersion  oil. 

9.  Several   ordinary  glass  tumblers,  some  of  which  are  used  for 

holding  platinum  needles,  clean  water,  scissors,  forceps, 
glass  rods,  etc.  A  drawer  is  necessary  in  the  table  to 
hold  clean  cover-glasses,  slides,  test  papers,  filter  paper, 
watch  glasses,  staining  dishes,  etc.,  when  not  in  actual 
use. 

The  following  stains  and  reagents  are  placed  in  the  aforementioned 
rack  for  ordinary  use  : — 

1.  Loffler's  solution  of  methylene  blue. 

2.  Watery  alcoholic  solution  of  gentian  violet. 

3.  Watery  alcoholic  solution  of  fuchsin. 

4.  ZiehPs  carbol  fuchsin,  or  Ehrlich's  anilin  water  fuchsin. 

5.  Ehrlich's  anilin  water  gentian  violet. 

6.  Roux^s  double  stain. 

7.  2  per  cent,  acetic  acid  in  water. 

8.  3  per  cent,  hydrochloric  acid  alcohol. 

§  III.  Methylene  blue  and  gentian  violet  give  excellent  staining 
results,  and  are  useful  stains  for  most  bacteria,  while  fuchsin  is  one  of 
the  simplest  and  most  rapid  stains  to  manipulate.  ZiehPs  carbol 
fuchsin  is  the  most  stable  preparation  of  fuchsin  used  in  staining 
Tubercle  bacilli.  EhrlicK's  anilin  water  fuchsin  and  gentian  violet 
give  excellent  staining  results,  but  are  very  unstable,  soon  decomposing, 
requiring  to  be  freshly  prepared  every  three  weeks.  Roux's  double 
stain  is  specially  adapted  for  the  detection  of  Diphtheria  bacilli,  inas- 
much as  the  bacilli  stain  more  readily  and  with  greater  intensity  than 
any  of  the  other  organisms  usually  found  associated  with  them.  (For 
the  preparation  of  the  above  stains  and  reagents  see  §§  49-60.) 


BACTERIOLOGICAL  TECHNIQUE 


15 


§  IV.  GENERAL  METHODS  OF  BACTERIOLOGICAL 
INVESTIGATION. 

THE  MICROSCOPE. 

For  bacteriological  investigation,  a  modern  microscope  with  an 
Abbe  condenser,  iris  diaphragm,  and  low  and  high  power  lenses,  is 
necessary.  The  following  makers  can  be  selected  from  : — 


ZEISS. 

Objective  AA. 
DD. 

Homogenous  Immersion,  one-twelfth. 
Eye-pieces  II.  and  IV. 


LEITZ. 

Objective  III. 
VII. 

Homogenous  Immersion,  one-twelfth. 
Eye-pieces  I.  and  III. 


The  low  power  is  used  with  a  narrow  diaphragm  for  the  examina- 
tion of  colonies  of  bacteria  developed  on  plate  or  Petri  dish  cultures. 

Unstained  specimens  are  always  examined  with  a  narrow  diaphragm, 
whilst  for  stained  specimens  an  open  diaphragm  is  necessary.  In 
examining  double-stained  sections  an  open  diaphragm  is  used  for  the 
bacteria  and  a  narrow  diaphragm  for  the  tissue.  The  oil  immersion 
lens  after  use  must  be  cleaned  with  benzine  and  wiped  with  a 
chamois  skin  or  special  lens  paper.  Excess  of  immersion  oil  is 
removed  from  the  cover-glass  with  xylol,  which  can  also  be  used  in- 
stead of  benzine  for  cleaning  the  lenses. 


§  V.    METHODS   OF   PREPARING   COVER-GLASS 
SPECIMENS. 

FLUIDS. 

The  platinum  loop,  previously  sterilized  in  the  Bunsen  flame,  is  used 
to  transfer  fluids  to  the  cover-glass. 

SEMI-SOLID  MATERIAL. 

A  drop  of  sterile  water  is  placed  on  a  cover-glass  and  the  material 
mixed  with  the  water  by  means  of  a  sterilized  platinum  wire  or  loop. 

TISSUES,  PIECES  OF  ORGANS,  ETC. 

Portions  of  the  material  are  spread  on  the  cover-glass  with  a 
sterilized  platinum  wire,  or  the  cover-glass  is  drawn  quickly  across 
the  cut  surface  of  the  tissue  or  organ. 


16  PRACTICAL  BACTERIOLOGY 

§  VI.  THE  HANGING  DROP. 

1.  Place  directly  in   the  middle  of  a  clean  cover-glass  with  the 
sterilized  platinum  loop  a  minute  drop  of  the  fluid  to  be  examined ; 
if  a  semi-solid,  a  drop  of  sterile  water,  physiological  salt  solution,  or 
bouillon  is  first  placed  on  the  cover-glass  and  then  inoculated. 

2.  The  edges  of  the  circular  cavity  in  the  hanging-drop  slide  are 
painted  with  a  narrow  strip  of  vaseline  with  a  cameFs-hair  brush. 

3.  Reverse  the  slide  and  place  it  upon  the  cover-glass  so  that  the 
inoculated   drop  is  exactly  in    the  centre    of   the   hollow   chamber. 
Apply  gentle  pressure  when  the  cover-glass  adheres  to  the  slide  by 
means  of  the  vaseline,  forming  an  air-tight  cavity.     The  preparation 
is  now  turned  upwards  quickly  (to  prevent  running  of  the  drop)  and 
examined  as  follows  : — 

The  convenient  method  of  beginners,  or  those  examining  many 
drop  cultures,  is  that  recommended  by  Nuttall,  consisting  of  making 
small  rings  upon  the  cover-glass  with  a  mixture  of  lamp-black  and 
blood  serum  applied  with  a  camePs-hair  brush  and  a  turn-table,  and 
passed  in  the  usual  manner  through  the  flame. 

1.  Cut  off  the  light  in  the  condenser  with  a  narrow  diaphragm 

about  the  size  of  a  pin-head. 

2.  Find  the  edge  of  the  hanging  drop  with  the  low  power  and  plain 

mirror. 

3.  Regulate  the  Abbe  condenser,  and  throw  the  light  into  the 

specimen. 

4.  Place  the  illumination  or  light  in  the  centre  of  the  specimen  by 

manipulating  the  mirror. 

5.  Screw  up  the  tube,  remove  the  low  power,  and  adjust  the  immer- 

sion lens. 

6.  Increase  the  size  of  the  diaphragm  to  about  the  size  of  a  pea. 

7.  Put  a  drop  of  immersion  oil  on  the  cover-glass. 

8.  Screw  down  the  tube  with  the  coarse  adjustment  until  the  point 

of  the  lens  touches  the  oil,  and  then  screw  back  the  tube 
without  breaking  the  oil  connection. 

9.  Screw  down  the  tube  again  by  means  of  the  coarse  adjustment 

until  the  object  comes  in  view. 

10.  Regulate  the  focus  with  the  fine  adjustment. 

11.  If  the  field  is  not  evenly  illuminated,  adjust  the  mirror  without 

further  removal. 

The  hanging-drop  method  is  used  to  study  the  morphobiological 
characteristics  of  organisms,  and  the  intravital  phenomena  connected 
therewith. 


BACTERIOLOGICAL  TECHNIQUE 


17 


§VII.   NUTTALL'S   MICROSCOPIC   THERMOSTAT. 

This  simple  apparatus  is  adapted 
for  use  with  different  microscopes 
that  are  placed  in  the  thermostat 
from  behind,  the  top  of  the  ap- 
paratus, which  is  slanting,  consist- 
ing of  two  laterally  movable  slides, 
the  inner  margins  of  which  are  made 
of  strips  of  felt  cut  according  to  the 
shape  of  the  microscope,  so  that  the 
tube  and  adjustment  remains  out- 
side. On  the  left  side  is  an  aper- 
ture large  enough  to  admit  the 
hand,  by  which  the  slide  can  be 
manipulated.  In  front  is  a  window 
admitting  the  light.  On  the  right 
side  screws  connected  with  the 
mechanical  stage  can  be  adjusted ; 
such  an  arrangement  is,  however, 
not  usually  necessary,  and  also  in- 
creases the  price.  The  thermostat 
is  closed  by  a  door  at  the  back. 
This  apparatus  is  useful  for  study- 
ing bacteria  or  protozoa  in  drop 
cultures,  and  has  also  been  found 
useful  in  studying  zooparasites. 


FIG.  6.— Nuttall's  Microscopic  Thermostat. 


§  VIII.  THE  COVER-GLASS  SPECIMENS. 
ORDINARY  METHOD. 

1.  Place  a  drop   of  sterile   water  on  a  clean   cover-glass  with  a 
sterilized  platinum  loop. 

2.  Inoculate  the  drop  with  a  small  quantity  of  the  material  under 
investigation — mixed  and  well  spread  with  the  platinum  loop  or  wire. 

3.  Allow  the  materialon  the  cover-glass  to  dry  in  the  air. 

4.  Fix,   by  passing  the  cover-glass,    preparation  side  uppermost, 
through  the  Bunsen  flame  three  times  at  intervals  of  one  second,  the 
cover-glass  being  held  with  '  Cornefs  Forceps.1 

5.  Stain  the  specimen  by  flooding  with  the  desired  filtered  stain, 

B 


18  PRACTICAL  BACTERIOLOGY 

allowing  it  to  remain  from  one  to  three  minutes.  (The  staining 
process  can  be  hastened  and  intensified  by  '  heating/  not  '  boiling,"* 
the  cover-glass  in  the  flame  until  vapour  arises.) 

6.  Remove   excess    of  stain   by  washing  the  specimen    with    the 
pipette  wash  bottle,  or  directly  under  the  tap.     (The  specimen  can  be 
examined  at  this  stage  by  laying  the  preparation  side  downwards  on 
a   slide   and    removing  excess   of  fluid   with  filter  paper,  and   if  of 
sufficient  interest  can  be  permanently  mounted.) 

7.  Remove  the  excess  of  water  by  blowing  with  or  without  a  glass 
tube,  and  dry.     (A  special  blower  for  this  purpose  can  be  made  from 
an  ordinary  atomizer  fitted  with  glass  tubes,  and  about  one-third  full 
of  calcium  chloride.) 

8.  Mount  in  xylol  balsam  and  examine  as  follows : — 

(1.)  Place  the  specimen  on  the  microscope  stage  so  that  about  the 
centre  of  the  cover-glass  lies  in  the  optical  axis. 

(2.)  Place  a  drop  of  immersion  oil  on  the  centre  of  the  cover- 
glass. 

(3.)  Screw  down  the  tube  with  the  coarse  adjustment  until  the 
point  of  the  lens  touches  the  oil ;  the  tube  is  now 
screwed  upwards  without  breaking  the  oil  connection. 

(4.)  Remove  the  diaphragm  below  the  Abbe  condenser,  and 
arrange  the  flat  mirror  so  that  the  field  is  somewhat 
lighted. 

(5.)  Screw  the  tube  carefully  down  with  the  coarse  adjustment 
until  the  preparation  comes  in  view. 

(6.)  Regulate  the  focus  with  the  fine  adjustment. 

(7.)  Obtain  the  maximal  illumination  by  manipulating  the  Abbe 
and  mirror  regulator. 

§  IX.   THE  CONTACT,  OR   IMPRESSION  SPECIMEN. 

1.  Lay  a  clean  cover-glass  gently  on  the  top  of  the  desired  colony 
on  the  plate  or  dish  culture,  apply  gentle  pressure,  and  lift  the  cover- 
glass  up  by  one  of  its  edges  without  lateral  movement. 

2.  Air  dry. 

3.  Fix  in  the  flame,  and  proceed  as  with  the  ordinary  cover-glass 
method,  process  No.  5,  §  8. 

The  above  specimens  differ  from  ordinary  cover-glass  preparations 
in  that  they  present  an  impression  of  the  organisms  as  they  were 
arranged  in  the  colony  from  which  the  preparation  was  made.  It  is 
important  to  note  that  '  liquefied-colonies '  cannot  be  used  for  the 
preparation  of  contact  or  impression  specimens. 


BACTERIOLOGICAL  TECHNIQUE  19 


§  X.  THE  GRAM   METHOD  OF  STAINING  COVER-GLASS 

SPECIMENS. 

1.  The  'fixed'  cover-glass  specimen  is  stained  two  to  five  minutes 
with  Ehrliclv's  anilin  water  gentian  violet  (see  §  55). 

2.  Wash  with  water. 

3.  Differentiate  with   Gram's   solution  of  iodine  (see  §  60)  until 
the  stained  surface  blackens   like  a  tea-leaf — which  usually  takes  a 
half  to  one  minute. 

4.  Decolorize  with  alcohol  until  no  more  stain  comes  away. 

5.  Wash  in  water. 

6.  Dry,  and  mount  in  xylol  balsam. 

The  Gram  method  is  employed  to  differentiate  Bacteria  into  two 
divisions : — 

(a)  Those  staining  according  to  the  method. 

(b)  Those  decolorized  according  to  the  method. 

According  to  Lehmann,  recent  researches  show  that  the  result 
depends  very  much  upon  the  age  of  the  culture  and  condition  of  the 
organism  at  the  time  the  specimen  is  stained,  as  well  as  the  method 
employed. 

The  Bacillus  of  Black  Quarter,  or  '  Symptomatic  Anthrax,1  can  also 
be  stained  under  certain  circumstances,  although  most  authors  write 
to  the  contrary. 

In  order  to  check  the  Gram  test  and  place  results  beyond  doubt, 
proceed  as  follows  : — 

Take  a  clean  cover-glass  and  put  a  small  quantity  of  the 
material  to  be  stained  on  one  half,  and  on  the  other 
half  a  small  quantity  of  a  young  culture  of  Bacillus 
anthracis.  Air  dry,  fix,  and  stain  by  the  ordinary  Gram 
method  above  mentioned,  so  that  both  materials  on  the 
cover-glass  are  thus  subject  to  the  same  reagents  and 
conditions. 

The  Bacillus  anthracis  staining  readily  according  to  the  Gram 
method,  one  can  thus  judge  whether  the  bacterial  species  under  in- 
vestigation stains  or  decolorizes  by  this  method. 

§  XL  THE  FOLLOWING  BACTERIA   STAIN   ACCORDING 
TO  THE  GRAM  METHOD. 

1.  Bacillus  anthracis. 

2.  Bacillus  of  Tuberculosis. 


20  PRACTICAL  BACTERIOLOGY 

3.  Bacillus  of  Leprosy. 

4.  Bacillus  of  Mouse  Septicaemia.  |  Different  varieties  of 

5.  Bacillus  of  Rouget,  or  Swine  Erysipelas,  j     the  same  species. 

6.  Bacillus  of  Tetanus. 

7.  Streptococci  of  Pyaemia  and  Erysipelas. 

8.  Staphylococcus  pyogenes  aureus. 

9.  Staphylococcus  pyogenes  citreus. 

10.  Staphylococcus  pyogenes  flavus. 

11.  Staphylococcus  pyogenes  albus. 

12.  Micrococcus  tetragenus. 

13.  Diplococcus  pneumoniae,  '  Fraenkel.' 

14.  Actinomyces  hominum.* 

15.  Actinomyces  bovis.* 

16.  Actinomyces  musculorum  suis. 

17.  Botryomyces  '  Bollinger.1  •(• 

18.  Discomyces  '  Rivolta.'  f 

19.  Botryococcus  ascoformans  '  Kitt.'  •(• 

20.  Bacillus  of  Diphtheria.  ^  Only  under  certain 
Bacillus  of  Black  Leg,  or  V       conditions     and 

'  Symptomatic  Anthrax.'     J      circumstances. 

21.  The  Lactic  Acid  Bacillus  of  Hueppe. 

§  XII.  THE  'CLADIUS'  CONTRAST  METHOD  FOR 
COVER-GLASS  SPECIMENS. 

By  adding  J  per  cent,  solution  of  picric  acid  in  water  to  1 
per  cent,  solution  of  methyl-violet  in  water  a  blue  indigo  colour  is 
the  result,  insoluble  in  water,  very  soluble  in  alcohol,  chloroform, 
anilin,  and  clove  oils. 

METHOD  OF  PROCEDURE. 

1.  Place  the  material  on  a  cover-glass  and  air  dry. 

2.  Pass  three  times  through  the  Bunsen  flame. 

3.  Stain  one  minute  in  a  1  per  cent,   watery  solution  of  methyl- 
violet. 

4.  Wash  in  water  and  dry  with  filter  paper. 

5.  Pass  through   J  per  cent,  picric  acid  solution  (see  §  61)  one 
minute. 

6.  Wash  in  water ;  dry  with  filter  paper. 

*  At  stage  4,  during  decolorization  with  alcohol  the  cover-glass  must  be  kept 
moving,  otherwise,  if  left  still,  the  Actinomyces  are  almost  entirely  decolorized. 
t  Named  according  to  investigators. 


BACTERIOLOGICAL  TECHNIQUE  21 

7.  Decolorize  in  chloroform  or  clove  oil,  and  repeat  the  applica- 
tion until  the  decolorization  is  complete.     '  Clove  oil  is  the  better  of 
the  two  for  permanent  specimens.1 

8.  Dry  and  mount  in  Canada  balsam,  dissolved  in  xylol. 
Result. — The  bacteria  are  stained  a  deep  indigo  blue. 

This  stain  has  very  little  affinity  for  fungi  cells  and  other  histo- 
logical  elements,  whilst  its  affinity  for  certain  bacteria  is  somewhat 
extraordinary.  The  bacteria  stained  by  the  Gram  method  also  stain 
by  this  method,  as  also  the  bacillus  of  malignant  oedema. 

§  XIII.  ZIEHL-GABBET  METHOD  OF  STAINING  TUBERCLE 
BACILLI  IN  COVER-GLASS  PREPARATIONS. 

1.  Spread  a  small  portion  of  the  material,  'sputum,  caseous  mass, 
mucus,  etc.,  that  is  most  likely  to  contain  the  bacilli,1  upon  a  clean 
cover-glass  in  as  thin  a  layer  as  possible.     4  Gunther  considers  it  is 
dangerous  in  making'  cover-glass  specimens  from  sputum  to  rub  two 
cover-glasses  together  and  then  separate  them,  as  the  caver-glasses  might 
break,  cutting  the  finger,  and  thus  inoculating  the  operator? 

(In  examining  the  sputum  of  suspected  tubercular  and  asthmatic 
patients  much  time  is  saved  by  using  the  sputum  attachment  of  the 
hand  centrifuge,  see  Fig.  7,  §  20.) 

2.  Air  dry,  and  fix  by  passing  three  times  through  the  Bunsen  flame. 

3.  Place  some  Ziehl-Nielsen's  carbol  fuchsin  (see  §  51)  in  a  watch 
glass  or  small  porcelain  evaporating  dish,  and  float  the  cover-glass  on 
the  surface,  material  side  downwards ;   heat  the  stain  until  vapour 
arises,  and  set  aside  one  minute  to  allow  the  stain  to  work.     '  Another 
method  is  to  flood  the  cover-glass  with  the  stain,  and  heat  three  or 
four  times  until  vapour  arises.1 

4.  Wash  the  cover-glass  in  water  to  remove  excess  stain. 

5.  Place  the  cover-glass  in   Gabbet's  solution  (see  §  52)  one  to 
three  minutes,  according  to  the  thickness  of  the  preparation  and  the 
intensity  of  the  fuchsin  stain. 

6.  Wash  in  water,  dry. 

7.  Mount  in  xylol  balsam. 

§  XIV.  METHOD  OF  STAINING  TUBERCLE  BACILLI  IN 
COVER-GLASS  SPECIMENS  WITH  EHRLICffS  ANILIN 
WATER  STAIN. 

This  method  is  recommended  by  Gunther. 

1.  Prepare  the  material  on  the  cover-glass  in  the  same  manner  as 
described  under  1  and  2  processes,  Ziehl-Gabbet  method,  §  13. 


22  PRACTICAL  BACTERIOLOGY 

2.  Place    some   Ehrlich's  anilin   water  fuchsin   or  gentian  violet 
(Ziehl's  carbol  fuchsin  can  also  be  used)  in  a  watch  glass  or  porcelain 
evaporating  dish,  float  the  cover-glass  on  the  surface,  material  side 
downwards  (if  it  sinks  it  does  not  matter),  heat  the  stain  until  vapour 
arises,  and  set  aside  one  minute  to  work. 

3.  Withdraw  the  cover-glass  with  the  small  forceps,  and  wash  off 
excess  stain  with  water. 

4.  Lay  the  cover-glass,  preparation  side  upwards,  in  a  watch  glass 
containing  3  per  cent.  HC1.  alcohol  (see  §.  70)  for  one  minute,  and 
move  to  and  fro,  when  it  is  decolorized. 

5.  Wash  with  water,  and  dry. 

6.  Stain  the  cover-glass  with  a  few  drops  of  weak  alcohol  or  watery 
solution  of  methylene  blue  or  malachite  green  for  fuchsin  stains,  and 
Bismarck  brown  for  the  violet  stain,  being  careful  not  to  stain  long 
enough  to  obliterate  any  stained  Tubercle  bacilli. 

7.  Wash  in  water,  and  dry. 

8.  Pass  through  the  Bunsen  flame.     '  If  passed  many  times  through 
the  flame  the  stained  bacteria  do  not  fade  so  readily.     Unna  employs 
this  method  for  Leprosy  bacilli.' 

9.  Mount  in  xylol  balsam. 

§    XV.   LOFFLEITS   METHOD   OF   STAINING   GLANDERS 
BACILLI   IN   COVER-GLASS  SPECIMENS. 

1.  Make  a  smear  preparation  from  a  young  nodule,  air  dry,  and 
fix. 

2.  Stain    in    Loffler's    methylene    blue    solution    (see  §   54)  five 
minutes. 

3.  Place  the  preparation  one  second  in  a  1  per  cent,  solution  of 
acetic  acid  in  water,  which  is  rendered  a  Rhine  wine  colour  by  the 
addition  of  a  few  drops  of  a  watery  solution  of  trapaolin  OO. 

4.  Wash  quickly  with  distilled  water. 

5.  Dry,  and  mount  in  xylol  balsam. 

'  The  addition  of'  the  trapaolin  OO  acts  as  follows:  The  cell 
plasma  is  entirety,  and  the  nuclei  partially  decolorized 
while  the  bacilli  retain  their  colour? 

§  XVI.   METHOD  OF  STAINING   COCCI   AND  GONOCOCCI 
IN   COVER-GLASS   SPECIMENS. 

1.  Prepare  the  cover-glass  specimen  in  the  ordinary  manner,  air  dry, 
and  fix. 

2.  Flood  the  specimen  with  concentrated  watery  solution  of  methy- 
lene blue. 


BACTERIOLOGICAL  TECHNIQUE  23 

3.  Heat  in  the  flame  until  vapour  arises. 

4.  Wash  in  water. 

5.  Dry  with  filter  paper. 

6.  Mount  in  xylol  balsam. 

§  XVII.  KNACK'S   METHOD   OF   STAINING   GONOCOCCI 
IN   COVER-GLASS   SPECIMENS. 

1.  Make  the  cover-glass  from  the  pus  in  the  ordinary  manner,  air 
dry,  and  fix. 

2.  Stain  with  methylene  blue. 

3.  Place  the  cover-glass  in  a  1  per  cent,  solution  of  argonin  four 
minutes. 

4.  Wash  and  dry  the  specimen. 

5.  Place  the  cover-glass  in  a  watery  solution  of  fuchsin  1-20  for  ten 
seconds. 

6.  Wash,  dry,  and  mount  in  xylol  balsam. 

§  XVIII.  NEISSEITS  METHOD  FOR  THE  DIFFERENTIAL 
DIAGNOSIS  OF  DIPHTHERIA  BACILLI  IN  COVER- 
GLASS  SPECIMENS. 

1.  Cover-glass  specimens  are  prepared  from  cultures  grown  on 
Loffler's  blood  serum,  at  34°  and  35°,  not  exceeding  36°  C. ;  the 
cultures  must  also  not  be  younger  than  nine  and  not  over  twenty  to 
twenty-four  hours'1  old ;  air  dry,  and  fix  the  specimen  in  the  flame. 

2.  Stain   three   seconds  with  Neisser's  No.  1  solution  (see  Stains, 
§58). 

3.  Wash  the  specimen  with  water. 

4.  Stain  three  to  five  seconds  with  Neisser's  No.  2  solution  pre- 
viously filtered  (see  Stains,  §  58). 

5.  Wash  in  water,  dry,  and  mount  in  xylol  balsam. 

Result. — The  body  of  the  Diphtheria  bacillus  is  stained  brown,  con- 
taining blue  granules — as  a  rule  two — one  at  each  end,  or  only  one  at 
one  end,  seldom  any  in  the  middle.  The  granules  are  oval,  and  never 
found  free.  In  other  organisms  resembling  the  Diphtheria  bacillus, 
these  are  round,  and  always  situated  at  the  end,  close  to  one  another. 

§  XIX.  UNNA'S  METHOD  OF  STAINING  FUNGI. 

Solution  A.  Concentrated  watery  solution  of  methylene  blue. 

„        B.  Unna's  glycerine-ether  mixture. 

1.  Place  the  suspected  material  (crusts,  etc.)  on  a  slide,  and  saturate 
with  acetic  acid. 


24  PRACTICAL  BACTERIOLOGY 

2.  Crush  the  material  with  a  second  slide  laid  crosswise. 

3.  Heat  over  the  flame  until  the  acetic  acid  cooks. 

4.  Separate  the  two  slides  and  dry  over  the  flame. 

5.  Remove  fatty  substances  with  ether. 

6.  Cover  the  prepared  surfaces  with  solution  A,  and  heat  over  the 
flame  until  vapour  arises. 

7.  Wash  with  water. 

8.  Decolorize  in  solution  B,  three  or  four  seconds. 

9.  Wash  with  water. 

10.  Dry  with  filter  paper  and  over  the  flame. 

11.  Mount  in  xylol  balsam. 

The  fungi  are  stained  a  deep  blue  colour,  while  epithelial  structures, 
owing  to  the  glycerine-ether  mixture,  appear  green.  If  this  differentia- 
tion is  not  desired,  the  decolorization  with  glycerine-ether  can  be 
dispensed  with. 

§  XX.  ORDINARY  METHOD  OF  EXAMINING  MILK  FOR 
BACTERIA  IN  COVER-GLASS  SPECIMENS. 

1.  Dip  a  sterilized  platinum  wire  into  the  milk,  and  draw  the  wire 
sharply  across  the  surface  of  a  clean  cover-glass. 

2.  Air  dry,  and  fix  in  the  flame. 

3.  Hold  the  cover-glass  between  the  fingers,  and  flood  the  prepara- 

tion side  with  some  sulphuric 
ether  to  remove  the  fat. 

4.  To  demonstrate  ordinary 
bacteria  stain  with  methylene 
blue,  as  it  does  not  colour 
the  background  so  intensely  as 
fuchsin  or  gentian  violet.  To 
demonstrate  Tubercle  bacilli, 
stain  the  specimen  according 
to  the  special  method  given 
on  page  21.  The  centrifugal 
machine  can  also  be  used 
(see  Fig.  7),  and  the  resulting 
sediment  examined,  the  same 
way  as  ordinary  milk. 

The  advantages  of  the  cen- 
trifugal     machine     are      that 
FIG.  7.— Aitmann's  Hand  Centrifuge.  sedimentation    can    be   carried 

out  very  quickly,  and  the  discovery  of  Tubercle  bacilli  rendered  easier. 


BACTERIOLOGICAL  TECHNIQUE  25 


§  XXI.  ROTITS  METHOD  OF  EXAMINING  BUTTER  FOR 
TUBERCLE  BACILLI  AND  OTHER  BACTERIA  IN 
COVER-GLASS  SPECIMENS. 

1.  About  two  to  four  grammes  of  the  suspected  butter  is  placed  in 
a  test-tube  with  a  small  spatula  or  a  glass  rod,  the  test-tube  filled 
about  three-quarters  full  with  water,  placed  in  a  water  bath  at  50°  C., 
and  heated  until  the  fat  is  thoroughly  melted. 

2.  Place  a  cork  in  the  test-tube  and  shake  well  a  few  times  in 
order  to  separate  any  Tubercle  bacilli,  etc.,  from  the  fatty  substance. 

3.  Place    the  test-tube   cork   downwards  in  the  warm  bath   for 
fifteen  minutes,  until  the  fat  is  again  thoroughly  dissolved. 

4.  Place  the  test-tube  in  a  cool  place,  so  that  the  butter  fat  again 
solidifies. 

5.  The  fluid  contents  are  either  placed  in  the  centrifugal  apparatus 
or  set  aside  until  the  sediment  forms. 

6.  Some  of  the  sediment  is  placed  on  a  cover-glass,   air   dried, 
slightly  heated,  laid  in  a  mixture  of  one  of  ether  and  three  of  alcohol, 
removed,  dried,  and  fixed  in  the  flame. 

7.  The  specimen  is  stained  according  to  the  ordinary  process  for 
Tubercle  bacilli  (see  §  14). 

'  To  isolate  bacteria  by  this  process  from  the  butter  a  sterilized 
platinum  loop  is  dipped  in  the  fluid,  and  gelatine  cultures  instituted.' 

§  XXII.    METHOD   OF   STAINING   CAPSULE   BACTERIA 
IN  COVER-GLASS  SPECIMENS. 

Treat  the  fixed  preparation  as  follows : — 

1.  Stain  with  2  per  cent,   gentian  violet  watery   solution,  and 

intensify  the   staining   action   by  heating   until   vapour 
arises. 

2.  Wash  with  water,  and  dry. 

3.  Decolorize  the  capsules  with  2  per  cent,  acetic  acid  in  water, 

six  to  ten  seconds. 

4.  Wash  with  water. 

5.  Mount  preferably  in  water,  as  the  high  refractive  index  of 

balsam  renders  the  capsule  less  visible. 

Fuchsin  can  also  be  used  in  place  of  gentian  violet. 

Many  species  of  bacteria  which  were  supposed  not  to  possess  a 
capsule  show  them  very  distinctly  by  this  method. 

It  is  of  especial  value  in  differentiating  the  capsule  of  Bacillus 
anthracis  according  to  4  Johne.' 


26  PRACTICAL  BACTERIOLOGY 


§   XXIII.   METHODS  FOR  STAINING  CILIA  OR 
FLAGELLA. 

LoFFLEiTs  METHOD,  MODIFIED  BY  GUNTHER. 

1.  Take  a  young  culture  grown  on  the  surface  of  agar-agar  media, 
and  make  a  hanging-drop  specimen,  examine  it  under  the  microscope, 
and  note  if  the  bacteria  are  motile. 

2.  Put  a  drop  of  distilled   water  on  a  clean  cover-glass  with  a 
sterile  platinum  loop,  inoculate  the  drop  with  a  little  of  the  culture 
material,  and  spread  gently  over  the  surface  of  the  cover-glass. 

3.  Allow  the  specimen   to   air   dry,  and  then  pass  three  times 
through  the  flame,  but  be  careful  not  to  heat  too  much. 

4  Filter  a  few  drops  of  Loffler's  mordant  (see  §  62)  on  the  cover- 
glass,  and  allow  it  to  remain  one-half  to  one  minute.  (Heat  is  un- 
necessary, as  it  only  yields  dirty,  unsatisfactory  specimens.) 

5.  Remove  the  mordant  with  a  fine  stream   of  water  from  the 
wash-bottle,   and   dry   the   cover-glass   in   the  ordinary  manner  by 
blowing,  etc. 

6.  Filter  a  drop  of  anilin  water  fuchsin  solution  on  the  surface 
of  the  cover-glass,  or  without  filtering  place  a  drop  of  fresh  watery 
alcoholic  solution  of  fuchsin  on  the  cover-glass  with  a  pipette,  and 
heat  the  cover-glass  over  the  flame  until  steam  arises,  remove,  and 
allow  the  warm  stain  to  remain  one  minute  on  the  cover-glass,  then 
wash  off  with  water. 

7.  Dry  quickly,  and  mount  in  xylol  balsam. 

The  addition  of  acids  or  alkali  to  the  mordant  are  now  no  longer 
considered  necessary,  and  Giinther  considers  heating  the  mordant 
unnecessary. 

§  XXIV.  BOWHILUS  METHOD  OF  STAINING  FLAGELLA 
AND  BACTERIA  SIMULTANEOUSLY  WITH  ORCEIN. 

1.  A  small  quantity  of  material  is  taken  from  the  surface  of  a  young 
agar-agar  culture  (previously  tested  by  '  the  Hanging  Drop '),  and  a 
suspension  made  in  boiled  distilled  water  in  a  test-tube. 

2.  Leave  the  tube  undisturbed  for  five  minutes,  then  place  one  drop 
of  the  bacteria-suspension  on  a  clean  cover-glass,  and  air  dry. 

3.  Fix  in  the  flame,  holding  the  specimen  between  the  fingers  to 
prevent  excessive  heating. 

4.  Pour  some  orcein  solution  (see  Stains,  §  59)  in  a  watch  glass, 
float  the  cover-glass,  preparation  side  downwards,  on  the  surface  of 


BACTERIOLOGICAL  TECHNIQUE  27 

the  solution,  and  heat  gently — do  not  boil — leaving  the  specimen  in 
the  solution  ten  to  fifteen  minutes. 

5.  Wash  the  preparation  in  the  ordinary  manner  with  water. 
Examine  in  water,  and  if  satisfactory  mount  in  xylol  balsam.  The 
advantage  of  examining  the  specimen  in  water  is  that  the  flagella 
appear  more  distinct  than  in  balsam,  and  if  too  faintly  stained,  the 
specimen  can  again  be  placed  in  the  orcein  solution,  and  the  process 
rt 


§  XXV.  THE  FLAGELLA  OF  THE  FOLLOWING  BACTERIA 
STAIN  BY  THE  ABOVE  METHOD. 

1.  Spirillum  choleras  Asiaticae. 

2.  Bacillus  typhi  abdominalis. 

3.  Bacillus  coli  communis. 

4.  Klein's  Bacillus  of  Swine  Fever. 

5.  Bacillus  subtilis. 

6.  Bacillus  violaceus. 

7.  Bacillus  fluorescens  liquefaciens. 

8.  Bacillus  prodigiosus. 

9.  Proteus  vulgaris. 

10.  Vibrio  Finkler-Prior. 

11.  Vibrio  Metschnikoff. 

12.  Vibrio  aquatalis. 

13.  Vibrio  Berolinensis. 

14.  Vibrio  Rugula. 

15.  Bacteria  in  hay  infusion. 

16.  Bacteria  in  oat-straw  infusion. 

17.  Infusoria  and  various  bacteria  in  canal  water. 

18.  Bacillus  tetani. 

19.  Bacillus  oedematis  maligni. 

20.  Bacillus  of  Symtomatic  Anthrax. 

§  XXVI.    METHODS   OF   STAINING   SPORES. 

Endospores  possess  a  firm  membrane  or  capsule  of  great  resistance 
towards  staining  reagents,  and  can  only  be  stained  with  certainty  on 
cover-glass  specimens. 

ORDINARY  METHOD. 

1.  Prepare  the  specimen  in  the  ordinary  way  (see  §  8),  air  dry, 
and  fix  in  the  flame  by  the  ordinary  method. 

2.  Float  the  cover-glass,  preparation  side  downwards,  on  a  watch 


28  PRACTICAL  BACTERIOLOGY 

glass  or  small  porcelain  evaporating  dish  of  Ehrlich's  anilin  water 
fuchsin  or  gentian  violet,  or  ZiehFs  carbol  fuchsin,  heated  until 
vapour  arises,  when  it  is  set  aside  to  cool,  and  allow  the  stain  to  work 
in  for  one  minute.  '  This  process  is  repeated  five  times.'1 

3.  Decolorize  with  3  per  cent.  HC1.  alcohol,  allowing  one  minute 
to  work  in  ;  the  spores  remain  stained. 

4.  Wash  the  cover-glass  with  water. 

5.  Contrast  stain,  with  methylene  blue,  if  the  spores  are  stained  with 
the  fuchsin  stain ;  if  with  the  violet  stain,  then  use  a  Bismarck  brown 
as  a  contrast. 

6.  Wash  in. water,  dry,  and  mount  in  xylol  balsam. 

Result. — With  juchsin,  the  spores  are  stained  red  and  the  bacilli 
blue ;  with  gentian  violet,  the  spores  are  stained  blue  and  the  bacilli 
brown. 

§  XXVII.— SPECIAL  METHOD  OF  STAINING   THE  ENDO- 
SPORES  OF  BACILLUS  ANTHRACIS. 

1.  Make   a   hanging-drop  culture   (see    method,   §   6)   from    the 
heart's  blood  of  an  inoculated  mouse. 

2.  Place  the  hanging  drop  in  an  incubator  from  twenty-four  to 
forty-eight  hours,  at  35°  C.,  until  spores  are  developed. 

3.  Remove  the  cover-glass  from  the  slide. 

4.  Air  dry. 

5.  Remove  the  vaseline  with  xylol. 

6.  Fix  in  the  flame  in  ordinary  way. 

7.  Proceed  as  at  process  No.  2.       (Ordinary  method  of  staining 
Spores,  see  §  26). 

§  XXVIII.  THE  EXAMINATION  OF  BLOOD  BY  COVER- 
GLASS  METHODS. 

1.  Handle  the  cover-glass  with  forceps  only,  as  the  heat  of  the 
hand  injures  the  specimen. 

2.  Place  an  extremely  small  drop  of  blood  on  the  cover-glass,  either 
directly  from  the  living  subject  or  with  the  platinum  loop. 

3.  Spread  the  drop  of  blood  by  placing  a  second  cover-glass  on  the 
top   of  the  first.      Avoid  pressure,  and  draw  the  two  cover-glasses 
apart  in  a  horizontal  direction. 

4.  Air  dry  under  a  glass  cover. 

5.  The   cover-glass    can   be   fixed    by    either    of    the    following 
methods : — 


BACTERIOLOGICAL  TECHNIQUE  29 

(a)  Exposure  to  a  temperature  of  110-115°  C.  in  hot-air  chamber 

for  a  few  minutes. 
(6)  Immersion  in  absolute  alcohol. 

(c)  Immersion  in  equal  parts  of  absolute  alcohol  and  sulphuric 

ether  for  thirty  minutes. 

(d)  Immersion  in  a  solution  of  formalin  for  five  minutes. 

(e)  According  to  Ehrliclv's    method,  on    a    metal    plate,    which 

is  heated. 

The  above  methods  of  fixing  are  specially  adapted  for  detecting 
corpuscular  changes,  etc.  For  ordinary  bacteria  proceed 
as  at  No.  4,  general  method,  §  8. 

6.  To  stain  for  bacteria,  use  the  ordinary  stains  for  that  purpose. 
The  special  stains  for  Malaria  are  described  under  'Malaria'  in 
Part  III. 

Precautions. — In  examining  specimens  of  blood  for  bacteria  an 
excess  of  haemoglobin  is  liable  to  exist,  which  interferes  with  the 
isolation  of  the  organism.  Remove  the  excess  before  fixing,  by 
placing  the  cover-glass  specimen  in  a  three  per  cent,  solution  of  acetic 
acid  for  a  few  seconds,  wash  with  distilled  water,  dry,  and  proceed  as 
above  at  No.  4,  general  method,  page  17. 

§  XXIX.  METHOD  OF   REMOUNTING  AND  RESTAINING 
COVER-GLASS  SPECIMENS. 

1.  Heat  the  slide  in  the  flame  of  the  Bunsen  to  warm  the  balsam ; 
when  it  is  melted  remove  the  cover-glass,  and  transfer  to  another  slide. 

2.  To  restain  the  cover-glass  specimen,  place  it,  after  removal  from 
the  slide,  in  xylol  for  twenty-four  hours,  which  is  changed  several 
times  to  ensure  thorough  removal  of  the  balsam. 

3.  The  cover-glass  is  now  placed  in  absolute  alcohol  to  remove  the 
xylol,  and  stained  according  to  the  method  desired. 

§  XXX.   NECESSARY  PRECAUTIONS  IN  MANIPULATING 
COVER-GLASS  SPECIMENS. 

1.  Never  handle   a   heated    cover-glass   with  cold  forceps,  as  it 
generally  breaks. 

2.  All  specimens   must  be   thoroughly    air-dried,    otherwise    the 
albuminoids  coagulate  under  the  influence  of  heat.     Some  bacteria, 
i.e.,  Bacillus  anthracis,  change  their  form,  break  up,  are  surrounded  by 
a  halo,  and  lose  their  affinity  for  the  stain. 


30  PRACTICAL  BACTERIOLOGY 

3.  During  the  process  of  fixing,  the  specimen  must  on  no  account 
be  scorched  in  the  flame,  otherwise  the  form  and  staining  properties 
of  the  bacteria,  etc.,  are  entirely  lost. 

4.  To  remove  immersion   oil  from   the  cover-glass  of  a  freshly 
mounted  specimen  absorb  the  most  of  the  oil  with  a  piece  of  filter 
paper,  then  the  remainder  can  be  removed  with  xylol  when  the  balsam 
sets. 


§  XXXI.  METHODS  OF  EXAMINING  SECTIONS  OF 
ORGANS  AND  TISSUES  FOR  THE  DETECTION 
OF  BACTERIA. 

There  is  no  method  of  demonstrating  bacteria  in  unprepared 
specimens.  All  tissues,  organs,  etc.,  must  be  cut  into  sections  to 
study  the  relative  position  of  the  bacteria  present  in  the  tissue; 
and  before  cutting  into  sections,  the  tissue,  organs,  etc.,  must  be 
thoroughly  fixed  and  placed  beyond  the  reach  of  cadaveric  changes 
as  follows : — 

1.  Cut  the  portion  of  organ  or  tissue  into  pieces  the  size  of  a  nut 

before  putrefactive  changes  can  take  place. 

2.  Place  the  squares  of  tissue  on   small   pieces   of  filter  paper, 

write  the  name  of  the  organ  or  tissue  on  the  side  of  the 
paper,  and  place  in  alcohol,  when  the  block  of  tissue 
becomes  firmly  fixed  to  the  filter  paper,  which  also  assists 
in  keeping  the  portion  to  be  hardened  above  in  the 
anhydrous  stratum. 

3.  The  blocks  of  tissue  remain  two  or  three  days  to  harden  in 

the  alcohol,  which  is  changed  several  times. 

4.  Pieces  of  kidney,  liver,  and  muscle,  after  remaining  in  alcohol 

two  or  three  days,  can  be  fastened  to  a  block  with  the 
following  preparation : — 

Gelatine       .         .     1  gramme. 
Water          .          .     2  c.  c. 
Glycerine     .  4  c.  c. 

Heat  and  dissolve  to  a  thick  consistency,  when  the  tissue 
is  fastened  to  the  block  with  the  above  adhesive  mixture, 
place  in  alcohol,  preparation  side  downwards,  and  in 
twenty-four  hours  the  gelatine  fixing  will  be  sufficiently 
hard  to  admit  of  the  tissue  or  organ  being  cut  into 
sections  with  the  microtome. 


BACTERIOLOGICAL  TECHNIQUE  31 

The  above  method  is  suitable  for  firm  tissues  and  organs,  but  deli- 
cate structures  must  be  imbedded  in  celloidin,  or  frozen,  before  they 
can  be  cut  into  sections. 

Formalin  can  also  be  used  for  hardening  tissues,  etc.,  but  the  pieces 
must  be  very  small,  as  formalin  lacks  the  power  of  great  penetration ; 
however,  small  pieces  of  tissue,  etc.,  can  be  laid  in  one  part  of  40  per 
cent,  formalin  and  ten  parts  of  water  for  six  to  eight  hours,  and  then 
placed  in  absolute  alcohol. 


§  XXXII.  METHOD  OF  EMBEDDING  IN  CELLOIDIN. 

1.  Transfer  the  hardened  tissue  or  organ  from  the  alcohol  into 
equal  parts  of  alcohol  and  sulphuric  ether  for  twelve  hours. 

2.  Transfer  the  tissue,  etc.,  into  a  solution  of  celloidin  made  with 
equal  parts  of  alcohol  and  sulphuric  ether  and  enough  celloidin  to 
form  a  thin  syrup ;  small  pieces  of  tissue,  etc.,  remain  in  this  solution 
two  days,  while  larger  pieces  remain  four  or  five  days. 

3.  Remove  the  piece  of  tissue   and    place  in    a  small  porcelain 
evaporating  dish,  pour  in  some  of  the  lower  or  thicker  solution  of 
celloidin  until  the  tissue  is  covered,  when  the  dish  is  set  aside  until 
the  celloidin  is  quite  hard ;  at  least  twenty-four  hours,  sometimes 
longer,  are  required. 

4.  Cut  the  celloidin  away  from  the  sides  of  the  porcelain  dish, 
place  the  imbedded  tissue  on  a  block,  and  fasten  with  a  little  celloidin 
solution.     Expose  the  block  to  the  air  until  the  celloidin  hardens — 
about  two  hours — and  then  place  in  80  per  cent,  alcohol,  and  keep 
until  required.     Absolute  alcohol  must  on   no  account  be  used,  as 
it  dissolves  the  celloidin. 


§  XXXIII.    METHOD  OF  PREPARING  TISSUES  FOR 
FREEZING. 

The  method  of  cutting  sections  with  the  freezing  microtome  is  con- 
ducted with  a  special  apparatus  sold  with  most  of  the  modern  micro- 
tomes, consisting  of  an  ether  spray  and  special  platform  for  holding 
the  block  of  tissue.  A  small  microtome,  manufactured  by  Jung  of 
Heidelberg,  known  as  the  Students'  Microtome,  is  specially  adapted  for 
frozen  objects  and  those  imbedded  in  paraffin,  being  fitted  with  a 
mechanical  knife-guide,  enabling  the  most  inexperienced  to  make 
serial  sections  without  difficulty  (see  Fig.  8). 


PRACTICAL  BACTERIOLOGY 


§   XXXIV.   BEFORE   FREEZING. 

All  traces  of  alcohol  must  be  removed  from  the  hardened  tissue  as 
follows : — 

1.  Place  in  a  1  per  cent,  solution  of  40  per  cent,  formalin  in  water 
for  two  hours.  Pieces  of  fresh  tissue  are  prepared  in  a 
similar  manner. 


FIG.  8. — Jung's  Students'  Microtome. 


A  piece  of  Joseph's  paper  or  ordinary  lens  paper  is  laid  on  the 
freezing  plate  of  the  microtome,  the  tissue  laid  on  top  of 
it,  and  frozen  with  the  ether  spray.  When  hard  it  is  cut 
into  sections,  and  the  cut  sections  placed  in  water. 


BACTERIOLOGICAL  TECHNIQUE 


33 


§  XXXV.  For  cutting  sections  of  tissue  embedded  in  celloidin  a 
microtome,  with  the  knife  working  at  an  angle,  is  necessary,  and  a  most 
serviceable  microtome  is  manufactured  by  Schanze,  Leipzig  (see  Fig.  9). 

It  is  important  to  remember  that  whatever  microtome  is  used  in 
cutting  tissues  embedded  in  celloidin,  botht  he  preparation  and  knife 
must  be  kept  continuously  wet  with  alcohol,  and  the  cut  sections 


Fin.  9. — Schanze's  Microtome. 

removed  from  the  knife  with  a  camePs-hair  brush,  into  60  or  70  per 
cent,  alcohol.     The  following  conditions  are  absolutely  necessary  : — 

1.  A  sharp  microtome  knife. 

2.  Hard  tissue. 

3.  The  tissue  firmly  attached  to  the  block. 

In  cutting  sections  of  tissues  for  the   demonstration  of  bacteria 
they  need  not  be  any  thinner  than  0'02  mm. 

§  XXXVI.  To  stain  bacteria  in  sections,  the  section  is,  with  few 
exceptions,  always  brought  directly  out  of  the  alcohol  into  the 
colouring  solution. 

1.  Sections  remain  longer  in  the  dye  than  cover-glass  specimens. 

2.  It  is  often  necessary  to  warm  the  staining  solution  either  in 

C 


o 


i 


PRACTICAL  BACTERIOLOGY 

the  incubator  or  with  a  spirit  lamp  or  Bunsen  burner, 
'  but  only  till  vapour  arises? 

3.  Decolorizing   processes    have   the   effect   of  decolorizing   the 

nuclei  and  rendering  them  obscure ;  to  prevent  this,  stain 
the  sections  first  with  lithio-  or  picro-carmine.  (See 
Giinther's  modification  of  the  Gram  method,  §  38). 

4.  To  reduce  or  prevent  the  bleaching  effects  of  alcohol  during 

decolorization,  add  to  the  alcohol  a  small  quantity  of 
the  stain  with  which  the  section  was  treated  in  the  first 
place. 

5.  In  clarifying  sections  do  not  use  clove  oil  for  anilin  dyes 

(except  in  the  Cladius  method),  as  it  has  the  power  of 
removing  the  stain.  L^se  oil  of  cedar,  and  best  of  all  for 
bacteria  is  xylol. 

§  XXXVII.   METHODS  OF  STAINING  BACTERIA  IN 
SECTIONS  OF  ORGANS  AND  TISSUES. 

/  WEIGERT'S   ORIGINAL  METHOD. 

1.  Remove  the  section  out  of  alcohol  into  water,  or  directly 
into  the  stain  one-half  to  one  minute. 

2.  Into  stain  '  methylene  blue '  one  to  two  minutes. 

3.  Wash  the  section  in  water. 

4.  Into  one-half  per  cent,  solution  of  acetic  acid  in  water  for 
one  minute. 

5.  Into  absolute  alcohol  thirty  seconds,  spreading  the  section 
well  out. 

6.  Again  in  absolute  alcohol  thirty  seconds. 

7.  Into  xylol  thirty  seconds. 


8.  Place  on  a  slide,  dry  with  filter-paper,  and  mount  in  xylol 
jbalsam. 

Anthrax  bacilli  can  be  detected  in  sections  by  this  method, 
as  also  many  of  the  bacteria  belonging  to  the  Septicsemise 
Haemorrhagicae  group  of  organisms,  but  care  must  be  used  not 
to  carry  the  acid  and  alcohol  treatment  too  far.  The  Glanders 
bacilli  can  also  be  demonstrated  in  sections  of  tissue  with  this  stain. 

§  XXXVIII.  THE  GRAM-GUNTHER  METHOD  FOR 
SECTIONS. 

1.  Remove  the  sections  into  water  for  two  or  three  minutes. 

2.  Into  a  solution  of  picro-carmine  two  to  five  minutes. 


BACTERIOLOGICAL  TECHNIQUE  35 

3.  Wash  the  section  in  four  or  five  changes  of  water. 

4.  Into  alcohol. 

5.  Into  Ehrlich's  anilin  water,  gentian  violet,  or   methyl  violet 
(see  §  55)  one  to  two  minutes. 

6.  Remove  section  into  Gram's  iodine  solution  (see  §  60)  for  two 
minutes,  and  spread  the  section  well  out. 

7.  Into  alcohol  thirty  seconds. 

8.  Into  3  per  cent.  HC1.  alcohol  (see  §  70)  for  ten  seconds. 

9.  Now  into  alcohol  many  times  until  a  maximal  decolorization 
is  obtained,  i.e.,  until  no  more  colouring  matter  comes  away. 

10.  Into  xylol  one-half  to  one  minute. 

11.  Jiemove  the  excess  of  xylol  with  filter-paper,  and  mount  the 
section  in  xylol  balsam. 

When  the  Gram  method  is  used  for  sections  without  Professor 
Giinther's  modification,  only  the  bacteria  are  stained,  but  by  first 
staining  with  picro-carmine  a  beautiful  double-stained  specimen  is 
the  result,  enabling  the  relation  between  the  bacteria  and  the  tissue 
to  be  distinctly  observed. 

The  Gram  method  does  not  stain  the  cells  except  plasma  or 
granule  cells,  which  are  sometimes  mistaken  for  clusters  of  micrococci. 
Some  of  the  epidermis  cells  also  stain,  and  liver  cells  decolorize  with 
difficulty. 

The  same  bacteria  are  stained  in  sections  by  the  Gram  method  as 
are  stained  in  cover-glass  specimens  (see  §  11). 

§  XXXIX.  THE  CLADIUS  'CONTRAST  STAIN'  FOR 
SECTIONS. 

1.  Stain  the  section  with  1  per  cent,  watery  solution  of  methyl 
violet  for  two  minutes. 

2.  Wash  in  water,  and  dry  with  filter-paper. 

3.  Into  picric  acid  solution  (see  §  61)  for  two  minutes. 

4.  Decolorize  in  clove  oil,  and  dry  with  filter-paper. 

5.  Into  xylol. 

6.  Mount  in  xylol  balsam. 

This  stain  acts  in  a  similar  manner  to  Gram's  method,  and  for 
many  bacteria  it  is  preferable.  For  organisms  stained  by  the  method, 
see  Gram's  method,  §11. 

The  original  paper  does  not  mention  the  method  of  applying  the 
clove  oil ;  but  in  conducting  and  testing  the  special  action  of  this  stain 
it  was  found  necessary  to  place  the  section  on  the  slide  during  the 
process  of  decolorizing  writh  the  clove  oil,  to  prevent  its  curling  up. 


36  PRACTICAL  BACTERIOLOGY 

§  XL.   EHRLICtTS  METHOD  OF  STAINING  TUBERCLE 
BACILLI  IN  SECTIONS  OF  TISSUE,  ORGANS,  &c. 

(a)  ALCOHOL  SECTIONS. 

1.  Place  sections  in  Ehrlich's  anilin  water  fuchsin*  (see  §  55),  and 
set  the  glass  dish  in  the  incubator  at  37°  C.  for  one  hour  and  a  half, 
and  at  room  temperature  twenty-four  hours. 

2.  Wash  in  water  five  minutes. 

3.  Into  3  per  cent.  HC1.  alcohol  one  minute,  and  move  round,  not 
allowed  to  simply  lie  in  the  alcohol. 

4.  Wash  in  water. 

5.  Contrast  stain  with  methylene  blue  or  Bismarck  brown. 

6.  Wash  in  water. 

7.  Into  alcohol  to  remove  water. 

8.  Into  xylol. 

9.  Mount  in  xylol  balsam. 

*Ehrlich's    anilin   water  gentian  violet  can  also  be  used,  or  Ziehl's  carbo 
fuchsin. 

(b)  FROZEN  SECTIONS. 

1.  Transfer  the  sections  from  the  salt  solution  into  80  per  cent, 
alcohol  for  five  minutes  to  harden ;  or, 

2.  Harden    in  corrosive  sublimate,  1   to   1000-1500  for  one-half 
to  one  hour,  and  wash  in  water,  and  proceed  as  above  at  process 
No.   1. 

§  XLI.  LOFFLER'S  UNIVERSAL  METHOD  OF  STAINING 

SECTIONS. 

1.  Bring  the  sections  out  of  alcohol  into  Loffler's  methylene  blue 
(see  §  54)  for  five  to  thirty  minutes. 

2.  Into  1  per  cent  acetic  acid. 

3.  Into  absolute  alcohol. 

4.  Into  xylol. 

5.  Mount  in  xylol  balsam. 

The  amount  of  time  the  section  remains  in  the  acetic  acid  solution 
will  depend  on  the  variety  of  organism  the  operator  is  working  with 
By  this  method 

Bacilli  are  stained       =  dark  black-blue. 
Cellular  constituents  =  blue. 
The  Protoplasm          =  light  blue. 
This  method  can  be  employed  for  almost  all  bacteria. 


BACTERIOLOGICAL  TECHNIQUE  37 

§  XLII.  WEIGERTS  METHOD  FOR  FIBRIN  OR 
BACTERIA  IN   SECTIONS. 

1.  Harden  section  in  alcohol. 

2.  Stain  in  Ehrlich's  anilin  water  gentian  violet  (see  §  55)  five 
to  fifteen  minutes. 

3.  Wash  the  section  in  6  per  cent,  solution  of  chloride  of  sodium. 

4.  Dry  the  section  with  filter-paper. 

5.  Place  in  a  1  or  2  per  cent,  watery  solution  of  iodide  of  potash 
for  two  or  three  minutes. 

6.  Dry  with  filter-paper. 

7.  Decolorize  in  the  following  solution  : — 

Anilin  oil,    .         .         .     2  c.  c. 
Xylol,          .         .         .     1  c.c. 

8.  Place  the  section  in  xylol  to  remove  the  anilin  oil. 

9.  Mount  in  xylol  balsam. 

§  XLIII.  LOFFLER'S  METHOD  OF  STAINING  THE 
GLANDERS  BACILLUS  IN  SECTIONS. 

1.  Stain  the  section  in  Loffler's  methylene  blue  (see  §  54)  for  a 
few  minutes. 

2.  Place  the  section  in  the  following  solution  : — 

Aqua  distilli,    .         .         .         .  10  c.  c. 
Coned.  Sulphuric  acid,      .         .     2  drops. 
5  per  cent.  sol.  of  Oxalic  acid,  .     1  drop, 
for  five  seconds. 

3.  Decolorize  and  dehydrate  in  absolute  alcohol. 

4.  Place  the  section  in  xylol. 

5.  Mount  in  xylol  balsam. 

§  XLIV.   ANOTHER  METHOD  OF  STAINING  THE 
GLANDERS     BACILLUS    IN    SECTIONS    OF    TISSUES. 

1.  Transfer  the  sections  from  alcohol  to  distilled  water. 

2.  Transfer  from  the  water  to  a  slide,  and  dry  thoroughly  with 
filter-paper. 

3.  Stain    with    a   few   drops    of  the    following   stain    for   thirty 
minutes  : — 

Carbol.  fuchsin,      .         .     10  c.  c. 
Distilled  water,      .         .  100  c.  c. 


38  PRACTICAL  BACTERIOLOGY 

4.  Remove  superfluous  stain  with  filter-paper,  wash  the  section 
three  times  with  3  per  cent,  acetic  acid,  and  do  not  allow  the  acid  to 
act  for  more  than  ten  seconds  each  time. 

5.  Remove  all  traces  of  the  acid  with  distilled  water,  absorb  the 
water  with  filter-paper. 

6.  Dry  the  section  with  gentle  heat  according  to  the  dry  method 
of  Unna  (see  §  46).     Clear  in  xylol,  and  mount  in  xylol  balsam. 


§  XLV.   KUHNE'S    METHYLENE    BLUE    METHOD    FOR 
STAINING   SECTIONS. 

1.  Bring  the  sections  out  of  alcohol  into  Kuhne^s  methylene  blue 
(see  §  56)  from  a  half  to  one  hour  (leprosy  sections  remain  longer). 

2.  Wash  quickly  in  water. 

3.  Wash  in  water  containing  1'5  to  £  per  cent,  hydrochloric  acid 
until  the  section  becomes  light  blue  in  colour. 

4.  Transfer  the  section  to  a  solution  of  lithium  carbonate,  prepared 
as  follows : — 

Concentrated  watery  solution  of  carbonate  of  lithia,  68  drops. 
Water  10  c.  c. 

5.  Place  the  section  in  clean  water  three  to  five  minutes. 

6.  Immerse  the  section  for  a  short  time  in  absolute  alcohol,  to 
which  a  little  methylene  blue  in  substance  is  added. 

7.  Rinse  the  section  completely  in  pure  anilin  oil. 

8.  Place    the    section  in    thymol    or   oil    of   turpentine    for    two 
minutes. 

9.  Place  in  xylol. 

10.  Mount  in  xylol  balsam. 

The  advantages  of  this  method  are  that  the  bacteria  are  not 
decolorized,  whereas  the  tissues  are  sufficiently  so  to  render  the 
bacteria  visible,  and  admit  of  the  use  of  contrast  stains. 

§  XLVI.    UNNA'S   <DRYr   METHOD1   FOR   SECTIONS. 

Many  bacteria  during  the  washing  process  become  entirely  de- 
colorized, and  to  prevent  this  result  the  dry  method  of  Unna  is 
employed  as  follows  : — 

1.  The  section  is   removed   from   the  staining   solution  directly 
into  water. 

2.  Spread  the  section  out  on  a  slide,  and  remove  excess  of  water 
with  filter-paper. 


BACTERIOLOGICAL  TECHNIQUE  39 

3.  The  slide  must  be  carefully  heated  over  the  flame,  'not 
cooked,1  until  the  section  is  dry,  when  it  is  allowed  to  cool  and 
mounted  in  xylol  balsam. 

Contrast  stains  by  this  method  are  of  little  use,  as  the  tissue  cells 
are  too  much  altered  by  the  heating.  Professor  Gunther  recommends 
the  'Dry  Method  of  Unna'  as  the  only  way  to  obtain  permanent 
specimens  of  tubercle  bacilli  in  sections. 

§  XLVII.    NECESSARY   PRECAUTIONS   IN   THE   MANIPU- 
LATION AND  STAINING  OF  SECTIONS. 

1.  When  staining  sections,  never  do  a  number  at  once  until  you 
have  tested  the  correctness  of  your  method  in  a  few  first. 

2.  In   the  enumeration   of  the  various   staining  methods  precise 
directions  as  to  time,  etc.,  have  been  given ;  it  is  well,  however,  to 
remember  that  these  directions  are  for  uniform  preparations  of  the 
stains,  solutions,  etc. 

3.  By  staining,  the  difference  in  length  of  bacteria,  however  slight, 
is  demonstrated,  also   the   extremely   characteristic   shape   of  some 
species. 

4.  By  staining,  an  insight  is  gained   into   micro-organismal   life 
existing  in  the  tissues,  while  double  staining  enables  the  bacteria  to 
be  distinguished  from  the  tissues  with  remarkable  precision.     It  is  an 
art  requiring  great  care  and  study,  invaluable  in  the  hands  of  those 
who  know  how  to  employ  it. 


§  XLVIII.  THE  FOLLOWING  ARE  THE  MOST  COMMON 
STAINING  FAULTS. 

1.  Portions  of  organs  left  too  long  before  being  put  in  alcohol 
commence  to  putrefy,  i.e.,  putrefactive  bacteria  and  fungi  gain  access, 
and  in  staining  sections  from  such  a  specimen,  for  a  definite  organism, 
the  putrefactive  bacteria  are  also  stained,  and  such  being  the  case, 
particular  attention  must  be  given  to  the  distribution  of  the  bacteria 
throughout  the  section,  as  putrefactive  bacteria  penetrates  the  organ 
or  piece  of  tissue  from  without,  their  numbers  are  therefore  found  to 
diminish  in  proportion  to  the  distance  from  the  outside  surface,  while 
the  inner  portions  are  usually  quite  free  from  putrefactive  bacteria. 

2.  Many    staining   solutions,   i.e.,   carmine,    hsemotoxilin  —  even 
distilled  water,  and  some  of  the  anilin  colours  often  contain  bacteria. 

3.  Faulty  staining  is  liable  to  occur  by  the  Gram  method,  when 


40  PRACTICAL  BACTERIOLOGY 

part  of  the  colouring  matter  may  be  deposited  on  the  surface  of  the 
sections.  The  use  of  too  strong  acids  sometimes  cause  rod  organisms 
to  break  up  into  beads,  when  they  are  liable  to  be  mistaken  for  a 
chain  of  cocci.  In  tissues,  extreme  caution  is  necessary  in  the  exam- 
ination of  plasma  or  granule  cells.  These  cells  under  the  action  of 
anilin  dyes  behave  directly  the  opposite  of  all  other  cells.  They 
generally  occur  as  large  flat  formations  of  the  outer  wall  of  vessels, 
consisting  of  a  nucleus  and  very  fine  grained  protoplasm.  It  is  only 
the  protoplasm  of  those  cells  that  stains,  the  nucleus  does  not,  there- 
fore it  requires  close  observation  to  find  it,  while  the  cells  contain  a 
deeply  stained  mass  of  granules,  strongly  resembling  a  colony  of 
micrococci.  They  are,  however,  distinguished  by  the  granules  being 
of  unequal  size.  The  nucleus  present,  when  sought  for,  and  the 
occurrence  together  of,  one  or  more  cells  of  the  same  size.  When  the 
section  is  stained  with  methylene  blue,  the  plasma  cells  are  stained  a 
deep  violet  colour.  Plasma  cells  also  sometimes  stain  according  to 
Gram's  method,  which  further  leads  to  their  being  mistaken  for 
micrococci.  Some  parts  of  the  epidermis  also  stain  by  Gram's 
method,  and  liver  cells  decolorize  with  difficulty. 


STAINS. 
§  XLIX.     STOCK   SOLUTIONS. 

1.  Concentrated  alcoholic  solution  of  fuchsin. 

2.  Do.  do.  do.         gentian  violet. 

3.  Do.  do.  do.          methyl  violet. 

4.  Do.  do.  do.          methylene  blue. 

In  preparing  the  above  stock  solutions,  use  about  half  an  ounce  of 
each  dye,  and  place  in  8-ounce  bottles  with  glass  stopper,  filled  with 
alcohol.  These  solutions  are  not  directly  employed  for  staining 
purposes. 

§  L.     ORDINARY   STAINS   FOR   DAILY   USE. 

(1)  Take  of  the  undissolved  dye  2  grammes,  and  distilled  water 
85  c.  cm. 

Boil  five  to  ten  minutes,  and  after  cooling,  add  15  c.  cm.  of 
90  per  cent,  alcohol  ;  mix  thoroughly  and  filter. 

Or  (2)  mix  5  c.  cm.  of  the  concentrated  alcoholic  solution  of  the 
dye  desired  with  50  c.  cm.  of  distilled  water. 


STAINS  41 

Or  (3)  fill  an  ordinary  test-tube  three-quarters  full  of  distilled 
water,  and  add  enough  of  the  concentrated  alcoholic  or  watery  solution 
of  the  dye,  until  you  can  just  see  through  the  solution,  which  is  ready 
for  use. 

§  LI.     SPECIAL   STAINS   FOR   COVER-GLASS   SPECIMENS 
AND   SECTIONS   OF  TISSUES. 

ZiehPs  carbol.  fuchsin. 

Fuchsin        .  .  .  .  1*0  gramme. 

Acid,  carbolic  .  .  .  5*0  grammes. 

Alcohol       ....  lO'O  c.  cm. 

Aquadistilli  .         .  .  .  100     „ 

§  LII.     GABBETS  SOLUTION. 

Methylene  blue  .  .  .  1  to  2  grammes. 

Aqua  distilli       .  .  .  75  c.  cm. 

Concentrated  sulphuric  acid        .  25     „ 

This  solution  is  used  in  the  Ziehl  Gabbet  method  for  demon- 
strating the  presence  of  the  bacillus  of  tuberculosis ;  the  acid 
decolorizes,  whilst  the  methylene  blue  acts  as  a  contrast  stain,  and 
is  of  especial  value  owing  to  the  simplicity  and  rapidity  of  its  action. 

§  LIIL     ROUX'S   DOUBLE   STAIN. 

Dahlia  or  gentian  violet        .  .  *5  grammes. 

Methyl  green  .  .  .  1*5         „ 

Aqua  distilli  .  .  .          200  c.  cm. 

METHOD  FOR  COVER-GLASSES. 

1.  Air  dry  and  fix  cover-glass. 

2.  Stain  from  five  to  ten  seconds. 

3.  Wash  in  water. 

4.  Dry  and  mount  in  Canada  balsam  dissolved  in  xylol.     Sections 
remain  in  this  stain    twelve   hours,   then   washed,  dehydrated,  and 
mounted.     This  stain  is,  as  already  mentioned,  of  especial  value  in  the 
examination  of  diphtheritic  specimens. 

§  LIV.     LOFFLER'S  METHYLENE  BLUE. 

Concentrated  alcoholic  Solution  of  methylene  blue,  30  c.  cm. 

Watery  solution  of  caustic  potash  (1  : 10,000)       100     „ 

This  stain  is  specially  adapted  for  staining  the  glanders  bacillus. 


42  PRACTICAL  BACTERIOLOGY 

§  LV.     EHRLICITS  ANILIN  WATER,  GENTIAN   VIOLET, 
FUCHSIN,  OR  METHYL  VIOLET. 

Anilin  oil         .  4c.  cm.  )rrn.    .    ,  ... 

T..  ..,,-,  ^rvr,  r  Inis  is  known  as  amlm  water. 

Distilled  water       100     „      J 

Shake  the  above  well  together,  filter,  and  add  lie.  cm.  of  a  con- 
centrated alcoholic  solution,  gentian  violet,  methyl  violet,  or  fuchsin, 
whichever  stain  is  required ;  shake  the  mixture,  and  set  aside  from 
twelve  to  twenty-four  hours  before  using. 

These  stains  yield  good  results  in  staining  many  forms  of 
organisms,  especially  tubercle  bacilli,  and  in  conjunction  with  Gram's 
method ;  but  they  have  one  drawback,  they  are  very  unstable,  and 
soon  decompose,  requiring  to  be  renewed  every  three  weeks. 

§  LVI.    KUHNE'S  METHYLENE  BLUE. 

Methylene  blue       .  .  .  1-5  grammes. 

Absolute  alcohol     .  .  .  lO'O         „ 

5  per  cent,  acid  carbolic  in  water    .         100*0          „ 
Mix  the  methylene  blue  and  the  alcohol,  rub  in  a  mortar  until  the 

methylene  blue  is  thoroughly  dissolved,  and  then  add  the  5  per  cent. 

solution  of  carbolic  acid. 

§  LVII.     CHLOROFORM  FUCHSIN  SOLUTION. 

(1)  One  or  two  crystals  of  fuchsin  are  dissolved  in  chloroform 
2  to  3  c.  cm. 

Or  (2)  concentrated  alcoholic  solution  of  fuchsin,  three  or  four 
drops  chloroform  2  to  3  c.  cm. 

Arenas  method  of  staining  tubercle  bacilli  in  fatty  substances,  milk, 
etc.,  etc. 

§    LVIII.     NEISSER'S    STAINS    FOR    DIFFERENTIAL 
DIAGNOSIS   OF   DIPHTHERIA. 

I. 

Methylene  blue             ...  1  gramme. 

96  per  cent,  alcohol     .              .              .  20  c.  cm. 
When  dissolved,  add — 

Aqua  distilli    ....  950  c.  cm. 

Glacial  acetic  acid       .             .             .  50     „ 

II. 

Vesuvin  .  .  .  .8  grammes. 

Aqua  distilli  boiled      .  .  .     lOOO'O  c.  cm. 


MORDANTS  43 

§    LIX.     BOWHILL'S    ORCEIN     STAIN     FOR     FLAGELLA 
AND   BACTERIA. 

A  saturated  alcoholic  solution  of  orcein  (this  solution  possesses 
greater  staining  powers  if  allowed  to  stand  ten  days  before  use). 

A  20  per  cent,  solution  of  tannic  acid  in  water  (dissolved  by 
heating  before  use.  The  above  solutions  are  mixed  as  follows  with 
distilled  water  : — 

Saturated  alcoholic  solution  of  orcein    .  15  c.  cm. 

20  per  cent,  watery  solution  of  tannic  acid         10     „ 
Distilled  water  ....  30     „ 

Mix  and  filter. 

MORDANTS. 

For  Demonstrating  certain  Bacteria. 
§  LX.  GRAM'S  SOLUTION  OF  IODINE. 

Iodine  crystals,    ....  1  gramme. 

Iodide  of  potash,  .  .  .  2         „ 

Distilled  water,  .  .  .  300  c.  cm. 

§  LXI.  M.  CLADIUS'S  METHOD. 

One-half  per  cent,  solution  of  picric  acid  in 

distilled  water,     .  .  .  50  c.  cm. 

Distilled  water,   .  .  .  .  50       „ 

Used  with  a  1  per  cent,  watery  solution  of  methyl  violet. 

For  the  Demonstration  of  Cilia  or  Flagella. 
§  LXIL  LOFFLER'S  MORDANT. 

Dissolve  by  heating  together 

Tannin,  .....  2  grammes. 

Distilled  water,  .  .  .  .  8  c.  cm. 

And  to  this  solution  add 

Concentrated   cold   water   solution    of   ferrous 

sulphate,  .  .  .  5  c.  cm. 

And 

Concentrated  alcoholic  solution  of  fuchsin,  1  c.  cm. 

Shake  well  together,  and  after  filtering,  it  is  ready  for  use. 


44  PRACTICAL  BACTERIOLOGY 

§  LXIII.  BUNGE'S  MORDANT. 

Ferric  chloride  B.P.  solution  diluted  1  to  20  of 

water,       .  .  .  .  25  c.  cm. 

Saturated  aqueous  solution  of  tannin,     .  75     „ 

N.B. — Recent  researches  by  Gunther,  Fischer,  and  Lehman,  show 
that  the  addition  of  alkalis  or  of  acids,  according  to  Loffler,  are 
superfluous,  as  also  the  hydrogen  per  oxide  solution  added  by  Bunge 
to  his  mordant. 

§  LXIV.  SOLUTION  FOR  CLEANSING  AND  DISINFECTING 
—FOR  COVER-GLASSES. 

Newly  purchased  cover-glasses  are  placed  in  a  solution  of  nitric 
acid  and  water  for  twenty-four  hours ;  removed,  washed  in  water,  and 
kept  until  required  in  alcohol.  The  advantages  of  really  clean  cover- 
glasses  and  slides  are  appreciated  by  experienced  microscopists. 

§  LXV.  SOLUTION  FOR  DIRTY  COVER-GLASSES,  SLIDES, 
AND  OTHER  GLASSWARE. 

Water     .  .  .  .  50  c.  cm. 

Methylated  alcohol          .  .  45    „ 

Strong  ammonia  .  .  .  5    „ 

§  LXVI.  DISINFECTING  SOLUTION  FOR  POTATOES, 

HANDS,  &c. 

Bichloride  of  mercury     ...  1  gramme. 

Water     .....        1000  c.  cm. 
Hydrochloric  acid,  '  strong ' '     '  .  .  5     „ 

§  LXVII.  LOFFLEITS  METHOD  FOR  CLEANSING 
COVER-GLASSES. 

1.  Warm   the  cover-glasses  for   some  time  in  concentrated  sul- 
phuric acid. 

2.  Rinse  in  water. 

3.  Place  until  wanted  in  a  mixture  of  equal  parts  of  alcohol  and 
ammonia. 

4.  Before  use,  dry   with    a   cloth,  from   which  all   fat   has  been 
extracted. 


SPECIAL  REAGENTS  45 

SPECIAL    REAGENTS. 

§  LXVIII.  'SOLIDS.' 

1.  Boracic  acid.  7.  Acid,  oxalic.  13.  Sodium  causticum. 

2.  Celloidin.  8.  Potass,  causticum.    14.  Tannin. 

3.  Acid,  carbolic.  9.  Potassium,  Iodine.    15.  Zinc  (metallic). 

4.  Ferrous  sulphate.      10.  Picric  acid.  16.  Paraffin. 

5.  Iodine  crystals.          11.  Pyrogallic  acid.         17.  Grape  sugar. 

6.  Mercuric  bichloride.  1 2.  Sodium,bicarbonate.l8.  Milk  sugar. 

§  LXIX.  «  LIQUIDS: 

1.  Acetic  acid  (glacial).  6.  Ether,      sulphuric    11.  Acid,  nitric. 

anhydrous. 

2.  Alcohol,  absolute.      7.  Ferric  chloride.         12.  Acid,  sulphuric. 

3.  Ammon.  fort.  liqr.      8.  Formaline,  40  per  13.  Water,  distilled. 

cent,  solution. 

4.  Anilin  oil.  9.  Glycerine  (Price's).  14.  Celloidin. 

5.  Chloroform.  10.  Acid,  hydrochloric.  15.  Lysol. 

The  purposes  of  the  above  reagents  are  described  in  the  chapters 
dealing  with  specific  methods,  etc.,  etc. 

§  LXX.  DIFFERENTIATING  AGENTS. 

1.  Distilled  water ;    2.  Absolute  alcohol ;    3.  Acid  alcohol,  made 
as  follows  : — 

90  per  cent,  alcohol          .  .  .          100  c.  cm. 

Aquadistilli         ....         200     „ 
and  the  desired  percentage  of  either  hydrochloric,  nitric,  sulphuric, 
or  oxalic  acids. 

4.  Anilin  oil,  2  parts          .  .  ) 
Xylol,  1  part                                             } 

5.  Oil  of  cloves ;   6.  Gram  and  Cladius's  solutions  (see  §§  60,  61). 

§  LXXI.  SPECIAL  ACTIONS  OF  THE  ABOVE  REAGENTS 

1.  Distilled  water,  remove  surplus  stain. 

2.  Absolute  alcohol,  dehydrate  and  remove  stain. 

3.  The  acid  alcohols  are  used  to  decolorize  cover-glass  specimens 


46  PRACTICAL  BACTERIOLOGY 

and  sections,  in  order  to  differentiate  the  micro-organism  which  do 
not,  in  some  instances,  decolorize  as  readily  as  the  tissues. 

4.  The  anilin  oil  and  xylol  are  recommended  by  Weigert  in  his 
'  Fibrin,  or  Bacteria  Stain  in  Sections.1 

5.  Oil  of  cloves  is  used  in  the  Cladius's  method  for  sections. 

§  LXXII.  CLEARING  AND  MOUNTING  MEDIA. 

1.  Xylol. 

2.  Oil  of  cedar. 

3.  Turpentine. 

4.  Oil  of  origanum. 

5.  Canada  balsam  dissolved  in  xylol  is  the  best  mounting  medium. 

6.  Vaseline  for  preparing  hanging  drop  preparations. 


PART    II. 

THE    PREPARATION    OF    NUTRIENT 
MEDIA— BOUILLON    MEDIA. 

§   LXXIII.   ORDINARY  BOUILLON. 

1.  Cut  500  grammes  of  lean  beef  or  veal,  free  from  fat,  into  small 
pieces,  add  1  litre  of  distilled  water,  and  set  in  a  cool  place  for  twelve 
to  twenty-four  hours.     In  hot  weather  place  in  an  ice  chest,  or  boil 
over  an  open  flame  for  one  hour,  stirring  continually. 

2.  Place  the  mixture  in  a  clean  piece  of  cloth  or  fine  muslin,  strain 
into  a  flask,  and  squeeze  until  you  obtain  1  litre  of  meat  infusion. 
It  has  an  acid  reaction. 

3.  Add,  Peptone  siccum,  10  grammes. 

Common  salt,         5         „ 

4.  Heat  the  flask  in  the  water-bath  for  one  to  one  and  a  half 
hours,  or  warm  directly  over  the  flame,  using  an  ordinary  enamel 
sauce-pan   with  several  pieces  of  wire  gauze  or  a  sheet  of  asbestos 
intervening  between  the  flame  and  the  pan. 

5.  Make   slightly   alkaline  with  a  saturated  solution   of  sodium 
bicarbonate,  '  if'  too  much  alkali  is  added  the  bouillon  will  never  clarify? 
The  white  of  a  hen's  egg  can  be  added,  but  it  is  not  absolutely 
necessary. 

6.  Sterilize  in  the  steam  sterilizer  one  to  two  hours,  remove  and 
filter  through  two  thicknesses  of  filter-paper  when  '  it  is  cold?      If 
filtered  when  hot,  fatty  substances  pass  through,  '  it  must  be  filtered 
very  slowly? 

7.  Place  again  in  the  steam  sterilizer  for  one  hour,  and   if  on 
removal  the  bouillon  is  of  a  clear  golden  colour  '  it  is  good?     If  it  is 
'turbid"1  it  is  allowed  to  cool,  is  filtered,  sterilized  for  one  hour,  and 
the  process  repeated  until  it  is  thoroughly  clarified. 


48  PRACTICAL  BACTERIOLOGY 

8.  Pour  the  bouillon  into  clean  tubes  (about  10  c.  c.  in  each),  or 
else  into  small  Erlenmeyer  flasks,  and  close  with  cotton  wadding 
plugs.     (In  making  a  cotton  wadding  plug,  twist  the  cotton  together, 
and  when  placed  in  the  tube  the  relaxation  of  the  twist  ensures  a 
tight  Jit.)    ^ 

9.  The  tubes  or  flasks  are  finally  sterilized  in  the  steam  sterilizer 
for  fifteen  to  thirty  minutes  on  each  of  three  successive  days.     The 
bouillon  is  ready  for  use  if  on  cooling  it  still  remains  perfectly  clear. 


§  LXXIV.   GRAPE  SUGAR  BOUILLON. 

Put  half  a  gramme  of  grape  sugar  into  a  small  Erlenmeyer  flask,  add 
100  c.  cm.  of  bouillon  after  it  is  filtered,  and  proceed  as  in  process 
No.  7,  ordinary  bouillon,  §  73. 

MILK  SUGAR  BOUILLON. 

Prepare  the  same  as  grape  sugar  bouillon,  but  add  2  per  cent,  of 
milk  sugar  instead  of  \  per  cent,  of  grape  sugar. 


§  LXXV.  GLYCERINE  BOUILLON. 

Add  4  to  6  grammes  of  glycerine  to  1 00  c.  c.  of  bouillon  either  at 
process  No.  3  or  at  process  No.  7,  ordinary  bouillon,  §  73,  and 
continue  the  following  processes  : — 

Instead  of  using  lean  beef  or  veal  to  make  a  meat  infusion, 
Liebig's  Meat  Extract  can  be  substituted,  using  10 
grammes  of  the  extract  to  1  litre  of  distilled  water,  but 
the  resulting  medium  is  brown  coloured,  whereas  with  the 
meat  infusion  it  is  colourless. 


POTATO    MEDIA. 

§  LXXVI.  ORDINARY^  METHOD. 

1.  Select   some   good   potatoes   and   wash   thoroughly    with   the 
potato-brush  and  water,  cutting  out  any  eyes  with  the  potato-knife. 

2.  Lay  the  cleansed  potatoes  in  sublimate  solution,  (see  §  66),  for 
thirty  minutes. 

3.  Sterilize  in  the  steam  sterilizer  one-half  to  three  quarters  of  an 
hour. 


POTATO  MEDIA  49 

4.  Wash  the  hands  in  sublimate  solution,  remove  the  potatoes, 
and  cut  in  two  with  a  sterilized  potato-knife. 

5.  Lay  the  cut  pieces  of  potato  in  plate  culture  dishes  with  a 
piece  of  filter  paper  previously  soaked  in  the  sublimate  solution  in  the 
bottom  of  the  dish.     '  The  potatoes  are  not  used  until  cold.'' 

6.  Use  the  platinum  loops  to  inoculate  the  cut  surface  of  the 
potatoes,  and  spread  the  material  over  the  desired  amount  of  surface 
with  a  sterilized  potato-knife. 


§  LXXVII.  ESMARCITS  METHOD. 

1.  Thoroughly  wash  some  potatoes. 

2.  Peel  and  cut  off  the  ends. 

3.  Cut  into  sections  1  cm.  thick  and  place  in  clean  water. 

4.  Place  the  slices  in  Esmarch  dishes. 

5.  Sterilize  in  the  steam  sterilizer  three-quarters  of  an  hour. 

6.  If  the  Esmarch  dishes  are  not  sterilized  before  the  slices  of 
potato  are  put  in,  then  sterilize  on  each  of  three  successive  days  as 
follows : — 

First  day,  for  30  minutes  ^ 

Second  day,  for  15  to  20  minutes  \     In  the  steam 

Third  day,  for  15  to  20  minutes  J 


§  LXXVIII.     ROUX  AND   GLOBIG'S   METHOD. 

1.  Some  potatoes  are  thoroughly  washed,  the  ends  cut  off,  bored 
through  with  a  cork-borer,  and  the  cylindrical  pieces  of  potato  laid  in 
water. 

2.  The  potato  cylinders  are  cut  diagonally  in   two  and  placed 
again  in  water. 

3.  Take  some  ordinary  test-tubes,  place  some  cotton  wadding  or 
a  piece  of  glass  rod  in  the  bottom,  add  a  little  water,  put  a  piece  of 
potato  in,  and  shake  it  down  until  it  touches  the  material  in  the 
bottom  of  the  tube,  plug  the  tube  with  cotton  wadding. 

4.  Potatoes  as  a  rule  have  a  slight  acid  reaction,  and  to  render 
them  faintly  alkaline,  lay  the  potatoes  in  a  1  per  cent,  solution  of 
sodium  carbonate  before  placing  in  the  tubes  or  before  sterilizing. 

5.  Sterilize  on  each  of  three  successive  days  as  follows  : 

First  day,  for  30  minutes  ^ 

Second  day,  for  15  to  20  minutes  In  the  steam 

Third  day,  for  15  to  20  minutes        J          sterilizer. 

D 


.»*» 

50  PRACTICAL  BACTERIOLOGY 

§  LXXIX.     HOLZ'S   METHOD, 
POTATO  WATER  FOR  BACILLUS  TYPHI  ABDOMINALIS. 

1.  Wash  and  peel  500  grammes  of  potatoes,  grate  very  fine,  and 
squeeze  the  juice  through  a  linen  cloth. 

&  The  juice  is  left  twenty-four  hours  in  an  ice-chest  before 
filtering,  or  is  filtered  immediately  through  animal  charcoal. 

3.  Sterilize  one  hour  in  the  steam  sterilizer,  add  10  per  cent,  of 
gelatine  (Eisner  adds  1  per  cent,  of  iodide  of  potash  to  the  gelatine 
before  use),  sterilize  again  one  hour  in  the  steam  sterilizer,  and  fill 
into  test-tubes. 

4.  Sterilize  the  tubes  on  each  of  three  successive  days  as  follows  : — 

First  day,  for  30  minutes  "} 

a         jj        i     i  e  j.    on      •      i.          I      In  the  steam 
Second  day,  for  15  to  SO  minutes       >• 

Third  day,  for  15  to  20  minutes        J 

§  LXXX.     POTATO   WATER. 

FOR  CULTIVATING  BACILLUS  TUBERCULOSIS. 

1.  Grate  500  grammes  of  clean  peeled  potatoes,  add  500  c.c.  of 
water,  and  place  in  an  ice-chest  for  twelve  to  twenty-four  hours. 

2.  Decant  the  liquid  portion  and  add  enough  distilled  water  to 
make  1000  c.c. 

3.  Cook   one  hour  in  the  water-bath,  filter,  add  4  per  cent,  of 
glycerine,  and  fill  into  tubes. 

4.  Sterilize  the  tubes  on  each  of  three  successive  days  as  follows  : — 

First  day,  for  30  minutes  \ 

|       In  the  steam 


a          ij        *     i  *  j.    «A      •     * 

Second  day,  tor  la  to  xO  minutes 

Third  day,  for  15  to  20  minutes 


f         sterilizer. 


PEPTONE    MEDIA. 

§   LXXXI.     PEPTONE   WATER   SOLUTION,   'KOCH.' 

Witte's  peptone  .  .  .  .  2  parts. 

Sodium  chloride  ....  1  part. 

Distilled  water    .  .  .  .100  parts. 


MILK  MEDIA— EGG  MEDIA  51 

§  LXXXII.     DUNHAMS   PEPTONE   SOLUTION. 

Dried  peptone  ...  1  gramme. 

Sodium  chloride          .  .  .  0'5         „ 

Distilled  water  .  .  .100  c.c. 

Filter,  place  in  tubes,  and  sterilize  by  the  ordinary  discontinuous 
method  for  three  successive  days. 

This  solution  is  used  as  a  medium  to  determine  if  an  organism 
under  consideration  possesses  the  property  of  producing  indol  as  one 
of  its  products  of  nutrition. 

§  LXXXIII.     PEFIONE   ROSOLIC   ACID   SOLUTION. 

Dunharrfs  peptone  solution  .  .          100  c.c. 

and  2  c.c.  of  the  following  solution  :— 

Rosolic  acid  (Coralline)         .  .  0.5  gramme. 

Alcohol  (80  per  cent.)  .  .          100  c.c. 

are  boiled,  filtered,  placed  in  test-tubes,  and  sterilized  by  the  ordinary 
discontinuous  method  for  three  successive  days.  This  medium  is  used 
to  study  the  reactions  produced  by  different  bacteria. 


MILK    MEDIA. 

§  LXXXIV.  The  milk  must  be  quite  fresh  and  the  reaction  must 
not  be  acid.  Place  about  10  c.c.  in  each  tube,  plug,  and  sterilize  in 
steam  sterilizer  for  one  hour  on  each  of  three  successive  days.  When 
the  tubes  are  not  in  the  sterilizer  they  must  be  kept  at  about  20°  C. 

The  sterilization  of  milk  media  is  very  important,  as  the  spores  of 
some  of  the  milk  bacteria  are  most  resistant. 

§  LXXXV.  Milk  media  for  testing  the  reactions  of  certain 
bacteria  are  prepared  by  adding  a  few  drops  of  tincture  of  litmus  to 
the  medium  before  sterilization  until  it  is  slightly  blue  in  colour. 
Ordinary  milk  media  acquires  a  light  brown  colour  after  sterilization. 


EGG    MEDIA. 

§   LXXXVI.     HUEPPE'S  METHOD. 

1.  Wash  a  fresh  egg  with  a  brush  and  soap  and  water. 

2.  Sterilize   a   glass   dish    with    sublimate    solution,    wash    with 
sterilized  water,  and  dry  with  sterilized  wadding. 


52  PRACTICAL  BACTERIOLOGY 

3.  Make  an  opening  at  one  end  of  the  egg  with  a  hot  needle  and 
inoculate  the  egg. 

4.  Close  the  opening  in  the  egg  with  a  small  piece  of  silk  paper, 
and  then  apply  a  coating  of  collodion. 

§  LXXXVII.     GUNTHER'S   METHOD. 

1.  Wash  one  end  of  an  egg  with  soap  and  water  and  a  brush. 
£  Sterilize  one  end  of  the  egg  with  the  flat  surface  of  a  clean  hot 
potato-knife. 

3.  Heat  a  steel  needle,  and  when  cool,  make  an  opening  in  the 
sterilized  spot,   large   enough   to    admit  the   platinum  needle  with 
which  the  egg  is  inoculated. 

4.  Close  with  sterile  paper  and  collodion,  or  with  hot  sealing-wax. 

§  LXXXVIII.     EGG   ALBUMEN   METHOD. 

Take  the  white  of  an  egg  and  distribute  it  in  tubes,  slant,  and 
coagulate  as  with  blood  serum  (see  §  99.) 


GELATINE   MEDIA. 
§  LXXXIX.  NUTRIENT  GELATINE. 

1.  Make  1  litre  of  meat  infusion  (see  Bouillon,  processes  Nos.  1 
and  2,  §  73). 

2.  Gelatine  .  .  .  .100  grammes. 
Peptone  siccum .             .             .             .  10       „ 
Common  salt      .             .              .             .  5       „ 

are  placed  in  a  glass  boiling  flask,  and  the  meat  infusion  added  last. 

3.  The  gelatine  and  peptone  are  thoroughly  dissolved  in  a  water- 
bath,  between  40°  and  50°  C.,  or  directly  over  the  flame,  using  an 
ordinary  enamel  saucepan,  with  several  pieces  of  wire  gauze  or  a  sheet 
of  asbestos  intervening  between  the  flame  and  the  pan. 

4.  Test  the  reaction,  make  alkaline  with  a  concentrated  solution 
of  sodium  carbonate   (the  same  caution  is  requisite  as  described  at 
Bouillon,  process  No.  5,  §  73),  and  add  a  fresh  egg. 

5.  Place  in  the  steam   sterilizer  for  one  and  a  quarter  hours  (too 
long  boiling  lowers  the  solidifying  point  of  the  gelatine),  remove  and 
filter  through  two  thicknesses  of  filter  paper.      (Fold  the  filter  paper 
with  a  sharp  point  pushed  well  down  into  the  neck  of  the  glass  funnel^ 


AGAR-AGAR  MEDIA  53 

which  is  placed  obliquely  in  the  flask,  and  a  small  Bunsen  flame  fixed  at 
an  angle  a  short  distance  from  the  neck  of  the  glass  funnel.  The 
gelatine  Jilt  ers  easily  by  this  method,  and  a  hot  water  funnel  is  un- 
necessary.) 

6.  The  reaction  is  again  tested,  and  if  still  faintly  alkaline,  fill  into 
tubes,  about  10  c.c.  in  each,  and  close  with  cotton  wadding  plugs. 
(;  The  Koch  method  of  sterilizing  wadding  and  tubes  before  use  is  now 
dispensed  with,  except  for  blood  serum  media.'') 

7.  Sterilize  the  tubes  of  gelatine  in  the  steam  sterilizer  for  fifteen 
minutes  on  each  of  three  successive  days.     The  gelatine  must  finally 
be  as  clear  as  glass. 

§  XC.  GRAPE  SUGAR  GELATINE. 

To  100  c.c.  of  nutrient  gelatine,  before  it  is  sterilized  (see  §  89, 
No.  6),  add  2  grammes  of  grape  sugar. 

§  XCI.  MILK   SUGAR   GELATINE. 

Add  2  grammes  of  milk  sugar  to  100  c.c.  of  gelatine  in  the  manner 
described  for  grape  sugar  gelatine. 

§  XCII.  THREE  PER  CENT.  COMMON  SALT  GELATINE. 

Add  3  per  cent,  instead  of  the  J  per  cent,  of  common  salt  used  in 
the  ordinary  process  of  making  nutrient  gelatine. 


AGAR-AGAR    MEDIA. 

§  XCIII.  ORDINARY  AGAR-AGAR. 

1.  Make  1  litre  of  meat  infusion  (see  Bouillon,  processes  Nos.  1 
and  2,  §  73),  add 

1  per  cent,  of  peptone  siccum     .  .  10  grammes. 

J  „  common  salt          .  .  5        „ 

2.  Place  the  above  in  a  clean  flask,  heat  in  a  water-bath  one  to 
one  and  a  half  hours,  and  add  1  to  2  per  cent.  (1J   is  the  amount 
generally  used)  of  agar-agar,  finely  powdered  or  cut  into  very  small 
pieces,  and  boil  five  to  eight  hours  until  the  agar-agar  is  thoroughly 
dissolved. 

3.  Make  alkaline  with  sodium  bicarbonate  solution. 


54  PRACTICAL  BACTERIOLOGY 

4.  Place  in  the  steam  sterilizer  one  to  two  hours. 

5.  Filter  through  ordinary  filter  paper  or  flannel    in  the  steam 
sterilizer.* 

6.  Fill  the  desired  quantity  into  test-tubes,  and  close  with  cotton 
wadding  plugs. 

7.  Sterilize  in  the  steam  sterilizer  for  fifteen  minutes  on  each  of 
three  successive  days. 

§  XCIV.  GRAPE  SUGAR  AGAR. 

Add  half  a  gramme  of  grape  sugar  to  100  c.c.  of  agar-agar  medium 
after  it  is  filtered,  and  proceed  as  at  §  93,  No.  6.  In  filtering  grape 
sugar  agar  into  tubes  it  is  customary  to  fill  the  tubes  two-thirds 
full,  as  this  medium  is  used  in  the  investigation  of  anaerobic  and 
gas-forming  bacteria. 

§  XCV.  GLYCERINE   AGAR. 

Add  4  to  6  grammes  of  glycerine  to  100  c.c.  of  agar-agar  medium, 
either  before  or  after  filtering,  and  proceed  as  at  §  93,  No.  6. 

§  XCVI.  WURTZ'S  LACTOSE  LITMUS  AGAR. 

1.  To  ordinary  slightly  alkaline  agar-agar  add  2  to  3  per  cent,  of 
lactose. 

2.  Fill  into  tubes  and  sterilize  by  the  ordinary  method  for  three 
consecutive  days. 

3.  When   sterilization   is   complete,   add  enough  sterilized  litmus 
tincture  to  give  the  medium  a  decided  pale  blue  colour. 

Bacteria  causing  fermentation  of  lactose,  when  grown  on  this 
medium,  develop  into  colonies  of  a  pale  pink  colour  with  a  corre- 
sponding reddening  of  the  surrounding  medium.  Bacteria  incapable 
of  producing  fermentation  grow  in  pale  blue  colours  and  cause  no 
reddening  of  the  surrounding  media.  (Instead  of  agar-agar,  ordinary 
nutrient  gelatine  can  be  substituted  in  the  preparation  of  this  medium.) 

§  XCVII.  BLOOD  AGAR. 

1.  A  few  drops  of  human  or  pigeon's  blood,  obtained  under  sterile 
precautions,  are  spread  over  the  oblique  surface  of  some  ordinary 
sterile  agar-agar  tubes. 

*  The  author  filters  agar  through  two  pieces  of  moleskin  cloth  under  pressure. 


BLOOD  SERUM  MEDIA  55 

£  The  prepared  tubes  are  placed  in  the  incubator  at  37°  C ,  and 
the  non-sterile  tubes  placed  on  one  side.  (This  medium  is  specially 
adapted  for  the  gonococcus  and  Influenza  bacillus.) 


§  XCVIII.  AGAR-AGAR  GELATINE. 

This  medium  is  prepared  in  the  same  way  as  ordinary  agar-agar, 
(§  93),  using  1-5  grammes  of  agar-agar,  and  when  it  is  dissolved 
adding  50  grammes  of  gelatine. 


BLOOD    SERUM     MEDIA. 

§  XCIX.  FLUID  BLOOD  SERUM. 

1.  Collect  the  blood  from  a  living  animal  into  a  large  sterile  glass 
jar,  under  sterile  precautions,  and  close  it  tightly. 

2.  Place  the  jar  in  an  ice-chest  for  twenty -four  hours. 

3.  Remove  the  serum  into  tubes  with  a  sterile  pipette. 

4.  Place  the  tubes  of  serum  in  the  incubator  at  37°  C. 

5.  Remove  any  tubes  that  become  turbid. 

§  C.  SOLID  BLOOD  SERUM. 

1.  Collect  the  blood  the  same  as  in  No.  1  process  for  fluid  blood 
serum. 

2.  About  fifteen  minutes  after  it  has  begun  to  clot,  pass  a  sterilized 
glass  rod  between  the  clot  and  the  wall  of  the  jar,  breaking  up  the 
adhesions  that  prevent  the  clot  from  sinking. 

3.  Close  the  jar  tightly,  and  place  in  an  ice-chest  twenty-four  to 
forty-eight  hours. 

4.  Draw  off  the  serum  with  sterilized  pipettes  into  sterilized  tubes, 
and  close  with  sterilized  cotton  wool  plugs.     (This  is  the  only  medium 
where  the  tubes  and  plugs  are  sterilized  before  filling. ) 

5.  Place  the  tubes  in  a  blood  serum  sterilizer,  and  heat  to  65°  or 
68°  C.  for  one  hour  on  each  of  five  successive  days,  and  place  in  the 
incubator  at  37°  C.  for  several  days  to  test  whether  they  are  sterile. 

6.  Place  the  sterile  tubes  in  Koch's  apparatus  for  solidifying  blood 
serum  (see  Fig.  10,  p.  56),  and  heat  to  75°  or  80°  C.  until  the  serum  is 
solidified. 


56  PRACTICAL  BACTERIOLOGY 


§  CI.  BLOOD  SERUM  'QUICK  METHOD.1 

Processes  1  to  4,  the  same  as  §  100,  solid  serum. 

5.  Place  the  tubes  in  the  serum  sterilizer  and  heat  at  once  to  90° 
or  95°  C.  for  one  hour  or  more. 

6.  Place  the  tubes  in  an  inclined  position  in  the  steam  sterilizer 
for  one  hour  or  more.     The  temperature  must  not  run  too  high,  other- 
wise bubbles  form  in  the  serum. 

7.  Place  the  tubes  in  the  ordinary  wire  baskets,  and  sterilize  as 
usual  for  fifteen  minutes  on  each  of  three  successive  days. 

Serum  prepared  according  to  this  method  is  more  opaque  than 
when  prepared  according  to  method  §  100,  but  answers  all  practical 
purposes,  saves  time,  and  avoids  imperfect  sterilization,  somewhat 
common  under  the  former  method. 


FIG.  10. — Koch's  Apparatus  for  Congealing  Blood  Serum. 

§  CII.  LOFFLEITS  METHOD. 

Special  Media  for  Cultivating  the  Bacillus  Diphtherias. 

1.  Take  blood  serum,  3  parts ;  1  per  cent,  grape  sugar ;  Bouillon 
1  part.     Mix  well  together,  and  fill  into  test-tubes. 

2.  Solidify  between  60°  and  70°  C. 

3.  Sterilize  for  ten  minutes  in  the  steam  sterilizer  on  each  of  three 
consecutive  days. 

Dr  Nuttall  has  devised  a  bulb  for  the  collection  of  blood  serum. 
It  is  a  sterilizable  vessel  made  of  glass,  by  which  10  to  100  c.c.  of 
blood  can  be  collected,  and  under  proper  precautions  no  contamina- 
tion takes  place.  The  method  of  procedure  is  as  follows  : — 

Expose  a  femoral  or  carotid  artery,  and  adjust  two  ligatures; 
the  one  distant  from  the  heart  is  tightened  and  the  proximal  one  left 
loose  between  the  latter  and  the  heart.  The  artery  is  clamped;  a 
small  slit  is  now  made  in  the  wall  of  the  artery,  the  point  of  the  bulb— 
the  sealed  end  of  which  has  been  broken  off  and  rounded  in  the  flame — 
is  introduced,  and  the  artery  bound  tightly  around  it  with  the  loose 


MEDIA  FOR  NITRIFYING  BACTERIA  57 

ligature.  The  clamp  is  then  removed,  when  the  bulb  is  quickly  filled 
with  blood.  Replace  the  clamp,  remove  the  bulb,  and  seal  in  the  gas 
flame.  The  loose  ligature  is  now  tightened  and  the  wound  closed. 
The  glass  bulb  is  put  in  a  cool  place  until  coagulation  has  occurred. 
The  serum  is  withdrawn  with  a  sterile  pipette. 

§  CIII.    MEDIA  FOR  MOULD  FUNGI. 

1.  Place  some  dry  bread,  finely  grated,  into  test-tubes  about  1| 
inch  high  in  each  tube,  or  into  Erlenmeyer  flasks,  about  1  inch  in 
each. 

2.  Add  enough  water  to  convert  the  bread  into  a  paste,  and  close 
the  tubes  or  flasks  with  cotton  plugs. 

3.  Sterilize  in  the  steam  sterilizer  for  fifteen  minutes  on  each  of 
three  successive  days. 

The  bread  paste  having  an  acid  reaction  cannot  be  employed  for 
the  cultivation  of  bacteria. 

§  CIV.  SABOURAUD^S  MEDIUM  FOR  FAVUS,  ETC. 

Peptone  .  .  .  0'8  gramme. 

Mannite          .  .  .  3'80  grammes. 

Aqua  distillata  .  .         lOO'O  c.c. 

Agar-agar       .  .  .  1*40  gramme. 

Prepare  in  the  ordinary  manner,  make  slightly  alkaline,  fill  in 
tubes,  and  sterilize  fifteen  minutes  on  each  of  three  successive  days. 


MEDIA    FOR    NITRIFYING    BACTERIA. 

§  CV.  WINOGRADSKY'S  SOLUTION. 

Ammonia  sulphate  .  .  1  gramme. 

Potassium  phosphate       .  .  1        „ 

Pure  water          .  .  .       1000  c.c. 

Place  100  c.c.  of  the  above  solution  in  each  flask,  and  add  to  each 

0*5    to   1-0   gramme   of  basic   magnesium   carbonate,  suspended   in 

distilled  water,  and  sterilize  by  boiling. 

§  CVI.  PREPARATION  OF  TUBES,  FLASKS,  ETC.,  FOR 
THE  PRESERVATION  OF  CULTURE  MEDIA. 

Both  new  tubes  and  those  previously  used  should  be  boiled  for  an 
hour  in  a  2  to  3  per  cent,  solution  of  common  soda,  and  carefully 


58  PRACTICAL  BACTERIOLOGY 

swabbed  out  with  a  test-tube  brush,  using  the  cane  handle  form,  as 
wire  handles  are  apt  to  go  through  the  bottom  of  the  tube.  When 
thoroughly  cleansed,  rinse  with  a  warm  1  per  cent,  solution  of 
commercial  hydrochloric  acid  (this  is  to  remove  the  alkali),  rinse 
thoroughly  in  clean  running  water,  and  stand  top  down  until  the 
water  has  drained  from  them. 

Fill  the  desired  amount  of  media  into  the  tubes  from  a  small 
Erlenmeyer  flask,  or  use  a  funnel  with  a  pinch-cock,  prepared  for  the 
purpose  when  exactitude  is  necessary,  but  for  all  practical  purposes  an 
Erlenmeyer  flask  is  sufficient.  Care  must  be  taken  that  none  of  the 
material  is  dropped  in  the  inside  of  the  mouth  of  the  test-tube,  as  it 
will  cause  the  cotton  plugs  to  adhere.  The  filled  tubes  are  plugged 
with  cotton  wadding  plugs,  carefully  rolled  together  before  insertion, 
and  the  tubes  sterilized  in  the  steam  sterilizer  for  fifteen  to  twenty 
minutes  on  each  of  three  successive  days.  The  old  method  of  steriliz- 
ing the  tubes  and  cotton  wadding  before  filling  is  no  longer  in  vogue, 
the  discontinuous  sterilization  being  sufficient. 

§  CVII.  METHODS  OF  CULTIVATING  BACTERIA. 

1.  The  platinum  wires  and  loops,  both  before  and  after  use,  are 
sterilized  in  the  Bunsen  flame,  from  above  downwards,  the  wire  being 
held  almost  perpendicular  in  the  flame,  and  the  upper  portion  of  the 
glass   or  metal  rod  it  is  attached   to    passes    several    times   directly 
through  the  centre  of  the  flame. 

2.  Fluid  cultures  are  inoculated  with  one  platinum  loop- full  of  a 
pure  culture. 

3.  Gelatine  and  agar-agar  stab  cultures  are  made  with  the  platinum 
wire,  making  only  one   stab  in  each   tube,   reaching  almost  to  the 
bottom  of  the  medium. 

4.  A  gar-gelatine  and  potato  contact  cultures  are  made  with  the 
platinum  loop,   the   material    being  spread   over  the  surface  of  the 
media.     The  agar  and  gelatine  media  being  solidified  obliquely,  whilst 
the  potato  surface  is  either  oblique  or  flat,  when  the  material  can  be 
further  spread  with  a  potato-knife. 

§  CVIII.  KOCH'S  ORIGINAL  PLATE  CULTURE  METHOD. 

FOR  THE  ISOLATION  OF  GERMS  IN  A  PURE  CULTURE. 

1.  Take  three  tubes  containing  10  c.c.  of  sterilized  nutrient  gela- 
tine, and  melt  the  medium  by  heating  in  the  water-bath  at  40°  C. 

2.  When  the  first  tube  has  cooled  to  30°  C.,  remove  the  cotton 


CULTURE  METHODS 


plug,  and  heat  the  mouth  of  the  tube  in  the  Bunsen  flame,  inoculate 
with  a  platinum  loop  of  a  liquid  culture,  or  a  trace  of  any  semi-solid 
culture,  or  other  desired  material.  After  inoculation  distribute  the 
organisms  evenly,  allowing  the  fluid  to  flow  gently  backwards  and 
forwards,  avoiding  contact  with  the  cotton  plug,  as  too  much  agitation 
causes  air-bubbles  to  form.  The  tube  is  held  between  the  thumb 
and  first  finger  of  the  left  hand,  while  the  cotton  plug  is  placed 
between  the  first  joints  of  the  first  and  second  fingers  of  the  same 
hand.  This  is  now  known  as  the  original  tube,  and  marked  c  O.1 

3.  Take  another  tube  of  gelatine,  remove    the  plug,   placing   it 
between  the  second  and   third  fingers  of  the  left  hand,  sterilize  the 
mouth  of  the  tube,  place  it  alongside  the  original  tube,  and  with  a 
sterilized  platinum  loop  transfer  three  loops  of  the  gelatine  in  the 
original  tube  into  the  second  tube.     Replace  the  plug  in  the  original 
tube  and  set  on  one  side.     The  second  tube  is  known  as  No.  1  Dilu- 
tion, and  marked  '  I.' 

4.  The  third  sterilized  tube  of  gelatine  is  inoculated  with  three 
platinum  loops  from  the  second  tube,  or  No.  1  Dilution,  in  the  same 
manner,  and  is  known  as  No.  &  Dilu- 
tion, and  marked  '  II.'     (For  marking 

the  tubes  for  future  identification, 
yellow  or  blue  coloured  pencils  for 
writing  on  porcelain,  metal,  or  glass, 
will  be  found  very  convenient.) 

5.  Three  sterilized  glass  plates  are 
now    removed    from    the   plate   box, 
placed   one  above  the  other  on  the 
ground  glass  plate  of  the  plate  culture 
apparatus  (see  Fig.  11),  and  the  con- 
tents of  the  above   tubes  poured  in 
order  on  the  plates.     When  the  gela- 
tine is  thoroughly  set,  the  plates  are 

,          T  111  i  1 1  FIG.  11  — Koch's  Plate  Culture  Apparatus. 

placed  on  glass  benches  one  above  the 

other,  the  original  plate  being  at  the  bottom  in  a  plate  culture  dish. 
Instead  of  using  plates,  it  is  simpler  to  pour  the  gelatine  into  sterilized 
Petri-dishes.  The  prepared  plates  or  Petri-dishes  can  be  placed  in  the 
incubator  at  22°  C.,  or  left  to  develop  at  room  temperature ;  as  a  rule 
only  two  of  the  plates  are  fit  for  future  observation — the  original 
generally  containing  too  many  colonies.  When  finished,  sterilize  the 
platinum  wires,  and  place  the  empty  tubes  in  the  disinfecting  solu- 
tion. A  piece  of  filter  paper,  with  the  number  and  date,  is  sometimes 
placed  on  the  glass  benches  below  the  plates. 


60  PRACTICAL  BACTERIOLOGY 

§  CIX.  METHOD  FOR  HARD  SUBSTANCES,  FAVUS 
CRUSTS,  ETC. 

1.  Sterilize  a  large  watch-glass  in  the  open  flame  and  cover  it  up. 

2.  Sterilize  the  bottom  of  an  ordinary  clean  test-tube  in  the  open 
flame. 

3.  Place  a  small  quantity  of  sterilized  bouillon  on  the  watch-glass, 
add  the  material  to  be  investigated,  triturate  until  thoroughly  re- 
duced and  mixed  with  the  bouillon,  and  proceed  as  in  process  No.  2, 
previous  method. 

§  CX.     AGAR-AGAR   PLATE   METHOD. 

In  making  agar-agar  plate  cultures  the  fact  must  not  be  lost  sight 
of  that  this  medium  solidifies  at  39°  C. 

1.  Melt  three  tubes  of  agar-agar  at  90°  C.  (freshly  prepared  tubes 
must  not  be  used,  as  the  water  of  condensation  will  cause  all  the  develop- 
ing colonies  to  run  together). 

2.  Place  in  a  water-bath  at  40°  C.,  remove  and  inoculate  quickly, 
making  the  necessary  series,  O1 2. 

3.  Pour  the  contents  of  the  tubes  preferably  into  Petri-dishes,  or 
if  plates  are  used,  without  rims ;  place  a  spot  of  melted  sealing-wax  at 
each  corner  to  prevent  the  agar-agar  sliding  off  the  plates. 

§  CXI.     METHOD   WITH   ORDINARY   AGAR-AGAR 
IN   TUBES. 

1.  Take  three  freshly  prepared  tubes  of  oblique  surface  agar-agar — 
usually  called  c  agar-slants ' — with  plenty  of  water  of  condensation  in 
the  bottom. 

2.  Inoculate  only  the  water  of  condensation   and   transfer   the 
bacterial  suspension  to  the  water  contained  in  the  series  of  tubes, 
making  the  usual  number,  O1  2. 

3.  When  the  dilutions  are  completed,  allow  the  water  of  con- 
densation in  each  tube  to  flow  gently  over  the  oblique  surface  of  the 
agar  medium,  and  place  in  the  incubator  at  37°  C. 

§  CXII.     BLOOD   SERUM— AGAR  PLATE  METHOD. 

1.  Take  three  tubes  of  sterilized  agar-agar,  melt,  retain  fluid  at 
40°  C.  in  a  water-bath,  inoculate,  and  make  the  usual  dilutions,  O1  2. 


CULTURE  METHODS  61 

2.  Pour  the  contents  of  the  tubes  into  Petri-dishes,  and  add 
sterilized  liquefied  blood  serum  heated  to  40°  C.,  mixing  it  thoroughly 
with  the  agar  medium.  (This  method  is  specially  adapted  for  the 
growth  of  Diphtheria  bacilli  and  gonococci.) 

§  CXIII.     AGAR  STROKE  CULTURE  PLATE  METHOD. 

1.  Liquefy  some  tubes  of  agar-agar,  pour  into  Petri-dishes,  and 
allow  to  solidify. 

2.  Dip  a  sterilized  platinum  wire  in  the  suspected  material,  raise 
the  lid  of  the  Petri-dish  obliquely,  and  make  several  strokes  with  the 
platinum  wire  across  the  surface  of  the  agar-agar.     Instead  of  using 
dishes,  take  six  or  eight  tubes  of  sterilized  oblique  surface  agar-agar, 
and  make  strokes  on  the  surface  of  the  medium.     (The  above  methods 
are  only  used  when  a  quick  diagnosis  is  necessary.) 

§  CXIV.     ESMARCH'S   ROLL   CULTURE   METHOD. 

1.  Take  three  large  wide-mouthed  test-tubes  containing  10  c.c.  of 
sterilized  nutrient  gelatine. 

2.  Liquefy  the  gelatine,  and  inoculate  with  the  material  under 
investigation,  making  the  usual  dilution,  O1 2. 

3.  Push  the  cotton  plugs  well  down,  cut  off  any  ends,  and  put  on 
an  india-rubber  cap. 

4.  Cool  the  gelatine  by  twisting  the  tube  round  and  round  on 
its  own  axis  in  ice-water,  when  the  gelatine  will  adhere  to  the  inner 
walls  of  the  tube  in  a  thin  film.     Before  putting  the  tube  in  the  ice- 
water,  roll  the  gelatine  round  the  periphery  of  the  internal  portion  of 
the  cotton  plug ;  by  this  means  the  centre  of  the  plug  remains  free 
from  gelatine,  otherwise  an  air-tight  cavity  results. 

5.  To  remove  a  colony  from  a  roll  culture,  place  the  tube  under 
a  low  power  lens  (see  special  apparatus,  Fig.  12),  select  the  colony, 
colour  the  outside  of  the  tube  over  the  colony,  and  then  remove  to 
other  media,  etc.,  with  a  bent  platinum  wire,  the  stain  outside  acting 
as  a  guide. 

§  CXV.     BOOKER'S   ROLL   CULTURE   METHOD. 

A  much  better  method  than  the  preceding,  though  depending 
upon  the  same  principles,  is  that  recommended  by  Booker. 

1.  Place  a  block  of  ice  of  convenient  size  in  a  dish,  resting  upon 
a  towel  (which  prevents  it  slipping). 


62  PRACTICAL  BACTERIOLOGY 

2.  Take  a  plugged  test-tube  filled  with  warm  water,  lay  it  upon 
the  block  of  ice,  and  thereby  melt  a  groove  in  the  ice. 

3.  The  test-tubes  (best  filled  with  5  to  6  c.c.   of  medium)  are 
placed  in  the  groove  after  inoculation,  and  revolved  rapidly  with  the 
fingers  of  the  right  hand    in    one   direction    only.     The   left   hand 
holding  the  dish  regulates   the  obliquity  of  the  tube,  which  at  the 
beginning  of  the  rotation  should  have  the  cotton  plug  at  a  higher 
level.     The  media  should  not  come  in  contact  with  the  plug. 

This  method  can  be  used  also  for  agar,  but  the  tubes  must  be 
kept  slanted  for  about  twenty-four  hours,  otherwise  the  agar  will  not 
keep  its  position  in  the  tube.  After  a  certain  number  of  hours  the 
agar  in  the  vicinity  of  the  cotton  plugs  dries  and  adheres  to  the  glass  ; 
with  gelatine  no  such  difficulty  occurs.  Rubber  caps  are  not 
necessary. 

§  CXVI.     METHOD   OF   COUNTING  COLONIES   IN 
ROLL   CULTURES. 

For  counting  colonies  in  roll  cultures,  an  apparatus  designed  by 
Esmarch  (see  Fig.  12)  may  be  used ;  it  consists  of  a  test-tube,  holder, 


FIG.  12.— Esmarch's  Apparatus  for  Counting  Roll  Culture  Colonies. 

and  lens  attached  to  a  stand.  The  holder  contains  quadrangular 
apertures  of  different  sizes,  the  number  of  colonies  being  counted  in 
the  larger  or  smaller  apertures  depending  on  the  number  of  colonies  in 
the  culture.  A  simpler  method  consists  in  attaching  a  piece  of  paper, 
in  which  squares  have  been  cut,  to  the  tube  by  means  of  elastic  bands, 
and  using  an  ordinary  hand  or  watchmaker's  lens  (Nuttall).  The 
estimation  of  the  total  number  is  made,  as  with  plate  cultures,  by 


CULTURE  METHODS  63 

measuring  the  surface  of  the  glass  covered  by  the  media,  and  multiply- 
ing by  the  number  of  colonies  found  in  a  certain  square. 

Gelatine  plate  or  dish  cultures  are  kept  at  ordinary  room  tempera- 
ture or  in  the  incubator  at  22°  C.,  and  agar-agar  plates  at  37°.  In 
twenty-four,  forty-eight,  or  seventy-two  hours  they  can  be  examined 
with  the  naked  eye,  hand  lens,  or  microscopically  with  a  low  power 
lens. 

The  various  kinds  of  characteristic  growths  are  noted,  and  the 
number  of  colonies  counted  with  the  Wolffhligel  apparatus  (see 
Fig.  13). 


FIG.  13.—  Wolffhiigel's  Counting  Apparatus. 

§  CXVII.  METHOD  OF  OBTAINING  A  PURE  CULTURE 
FROM  A  COLONY  ON  A  PLATE  OR  DISH. 

1.  Examine  the  plate  under  the  microscope  with  a  low  power,  or 
use  a  hand   lens,  and   find   the  desired   colony,  always  selecting  an 
isolated  colony  when  possible. 

2.  Take  a  tube  of  sterilized  nutrient  gelatine,  sterilize  a  platinum 
wire,  and  carefully  remove  a  part  of  the  desired  colony  on  the  point 
of  the  wire. 

3.  Remove  the  plug  from   the  gelatine  tube,  holding  the   tube 
perpendicular,  mouth  downwards,  in  the  left  hand,  and  make  a  stab 
culture  in  the  middle  of  the  gelatine  medium,  extending  almost  to 
the  bottom  of  the  tube,  heat  the  neck  of  the  tube  in  the  flame,  and 
replace  the  plug ;  with  liquid  media  the  tube  is  held  slanting. 

METHOD  OF  KEEPING  GROWING  CULTURES  PURE. 

1.  Inoculate  the  cultures    into  fresh  medium   every  fourteen   or 
twenty-one  days. 

2.  Place  the  tube   containing  the  original   culture   between   the 
thumb  and  first  finger  of  the  left  hand,  and  place  the  tube  to  be 
inoculated  between  the  first  and  second  fingers  of  the  same  hand. 


64  PRACTICAL  BACTERIOLOGY 

3.  Remove  both  cotton  plugs,  and  hold  between  third  and  fourth 
and  fourth  and  fifth  fingers  respectively  of  the  left  hand. 

4.  Pass  a  sterilized  platinum  loop  into  the  original  tube  without 
touching  the  sides,  and  transfer  one  loop  of  material  into  the  second 
tube,  using  similar  precautions. 

5.  Heat  the  necks  of  both  tubes  in  the  flame,  and  scorch  both 
plugs  before  returning  them. 

§  CXVIII.  THE  QUANTITATIVE  PLATE  CULTURE 
METHOD. 

This  method  is  used  to  determine  the  number  of  bacteria  in  a 
given  quantity  of  Material.  Fluids  are  examined  as  follows  :— 

1.  Transfer  with  a  sterilized  capillary  pipette,    y^  c.c.  to  1  c.c. 
of  the  material  into  10  c.c.  of  liquefied  sterilized  gelatine,  and  mix. 

2.  Another  tube  containing   10  c.c.   of  liquefied  gelatine  is  in- 
oculated in  a  similar  manner. 

3.  Pour  the  contents  of  the  tubes  on  plates,  as  in  the  ordinary 
plate  method  (§  108),  or  into  Petri-dishes  previously  sterilized,  and  set 
on  one  side  for  from  twenty-four  hours  to  seven  days. 

When  the  colonies  have  developed,  the  exact  number  is  ascer- 
tained with  the  Wolff hugePs  Counting  Apparatus  (see  Fig.  13).  ''Roll 
cultures  can  also  be  used.'' 

'Solid  Material ''  is  first  reduced  in  a  sterile  mortar  (see  Favus 
Crusts  Method,  §  109,  process  No.  2),  with  5  or  10  c.c.  of  sterile 
bouillon,  or  physiological  salt  solution,  and  then  tested  as  above. 

§  CXIX.  METHODS  OF  CULTIVATING  ANAEROBIC 
BACTERIA. 

Anaerobic  organisms  are  characterised  by  their  inability  to  grow 
in  the  presence  of  oxygen,  and  many  devices  are  employed  for  the 
exclusion  of  oxygen  from  the  cultures. 

The  preparation  of  suitable  media  and  cultivation  of  anaerobic 
bacteria  require  skill  and  knowledge  of  bacteriological  technique. 

§  CXX.  KOCH'S  METHOD. 

Inoculate  a  gelatine  plate,  and  cover  the  inoculated  surface  with  a 
thin  piece  of  sterilized  isinglass;  organisms  growing  beneath  this 
plate  are  supposed  to  grow  without  oxygen. 


ANAEROBIC  CULTURE  METHODS  65 

§  CXXI.  HESSKS  METHOD. 

Pour  some  sterilized  oil  on  the  surface  of  a  gelatine  stab  culture, 
and  the  growth  developing  along  the  track  of  the  needle  is  supposed 
to  be  of  an  anaerobic  nature. 

§  CXXIL  LIBORIOUS^S  METHODS. 

a.  Fill  a  test-tube   three-quarters  full  of  gelatine  or  agar-agar, 
sterilize,  and  place  in  a  vessel  of  boiling  water  for  ten  minutes  to 
expel  all  air  from  it.     Cool  the  medium  rapidly  in  ice-water,  when 
FIG-  14-          between   30°  and  40°  C.   and   still   fluid,   inoculate, 
solidify  rapidly,  and  seal  up  the  tube  in  the  flame. 
The   anaerobic   bacteria   develop    only  in  the  lower 
layers  of  the  medium. 

b.  For  this  method  the  special  Laborious  tube 
is  necessary  (see  Fig.  14).  Hydrogen  gas  is  passed 
through  the  side  tube  by  means  of  the  hydrogen 
apparatus,  until  all  air  is  expelled  (see  Fig.  17, 
p.  67).  The  contracted  parts  at  the  neck  and  side 
of  the  tube  are  sealed  in  the  flame,  but  owing  to  its 
small  capacity  give  better  results  with  fluid  media. 
For  the  necessary  precautions  to  be  observed  in 

Liborious's  Tube  for  •         i       i  p   -i  nr, 

Anaerobic  Cultures.     USlllg  hydrogen,  S66  §    1&5. 

§  CXXIII.   FILENKEL'S   METHOD. 

1.  Prepare  a  tube  in  the  same  manner  as  for  a  plate  or  Esmarch 
roll  culture. 

2.  Replace  the  cotton  by  a  sterile  rubber  stopper  with  two  glass 
tubes  passing  through  it,  and  plugged  with  cotton  wadding  before 
sterilization.     The  tubes  on  the  outside  of  the  stopper  are  bent  at 
right  angles  to  the  long  axis  of  the  test-tubes,  and  slightly  drawn  out 
in  the  flame.     One  of  the  tubes  reaches  within  0'5  c.  of  the  bottom  of 
the  tube,  while  the  other  is  cut  off  level  with  the  inside  of  the  stopper. 
The  hydrogen  apparatus  (see  Fig.  ^  7,  p.  67)  is  attached  to  the  end  of 
the  longest  tube,  and  hydrogen  allowed  to  bubble  through  the  tube 
until  all  air  is  expelled  and  its  place  taken  by  the  hydrogen.     The 
hydrogen  must  be  passed  through  the  gelatine  at  least  five  minutes,  to 
ensure  that  all  oxygen  is  expelled.     The  drawn  out  portions  of  the 
tubes  are  sealed  in  the  flame,  and  the  protruding  end  of  the  rubber 
stopper  painted  round  with  paraffin. 


66 


PRACTICAL  BACTERIOLOGY 


Before  using  the  hydrogen  apparatus  test  the  hydrogen,  and  make 
sure  it  is  free  from  oxygen,  as  follows : — 

Fill  an  ordinary  test-tube  with  water,  close  the  mouth  with  the 
thumb,  invert  it,  and  place  its  mouth  under  water ;  remove  the  thumb, 
and  the  water  will  be  kept  in  by  atmospheric  pressure.  Conduct  the 
hydrogen  into  the  test-tube  by  means  of  a  piece  of  rubber  tubing, 
when  the  water  in  the  tube  will  be  replaced  by  gas.  Hold  a  flame 
near  the  mouth  of  the  test-tube,  and  if  no  explosion  occurs  the 
hydrogen  is  safe  to  use. 


FIG.  15. 


FIG.    16. 


Kitasato's  Bottle  for  Anaerobic  Cultures. 


Botkin's  Apparatus  for  Anaerobic  Plate  Cultures. 


§  CXXIV.  Kitasato  devised  a  special  flat  bottle  for  the  making 
of  anaerobic  cultures  (see  Fig.  15).  Kitasato  and  Wiel  also  suggest 
the  addition  of 

Formic  acid,     .         .     0*3  to  0'5  per  cent. 

Glucose,  .         .         .     1-5  to  2         „ 

or  blue  litmus  tincture  in   5   per  cent,   per  volume  to  the  culture 
medium  in  addition  to  an  atmosphere  free  from  oxygen. 

§  CXXV.  Botkin  has  devised  a  special  apparatus  for  anaerobic 
plate  cultures — see  Zeitschriftfilr  Hygiene,  1890,  §  385  (see  Fig.  16). 


§  CXXVI.  BUCHNER'S  METHOD. 

The  bacteria  are  developed  in  an  atmosphere  robbed  of  its  oxygen 
by  pyrogallic  acid.  Either  oblique,  roll,  or  stab  cultures  are  made  in 
a  test-tube  which  is  placed  inside  a  larger  tube,  having  a  brass  support 
for  the  bottom  of  the  smaller  tube  to  fit  into  (see  Fig.  18,  p.  67). 
1  gramme  of  pyrogallic  acid  and  10  c.c.  of  TVth  normal  caustic 


ANAEROBIC  CULTURE  METHODS 


67 


potash  solution*  are  put  in  the  larger  tube,  which  is  tightly  plugged 
with  an  india-rubber  stopper.  The  oxygen  is  quickly  absorbed  by  the 
pyrogallic  acid,  and  the  organisms  develop  in  the  remaining  con- 
stituents of  the  atmosphere — nitrogen,  small  amount  of,  Co2,  and  a 
trace  of  NH3. 

§  CXXVII.  KASPAREE'S  METHOD  FOR  LIQUID  MEDIA. 

A  special  flask  is  necessary  for  this  method,  with  a  small  tube 
terminating  in  a  bulb  blown  into  its  neck  about  f  ths  of  an  inch 
above  the  top  of  the  liquid  medium,  which  is  prepared  as  follows : — 

1.  Fill  the  flask  with  bouillon  almost  to  the  neck,  then  add  3  c.c. 


FIG.  11 


FIG.  18. 


Buchner's  Tube  for  Anaerobic 
Cultures. 


Kipp's  Hydrogen  Apparatus. 

of  liquid  paraffin,  and  sterilize  the  whole  in  the  steam  sterilizer  in  the 
usual  manner.  This  expands  the  bouillon,  causing  the  paraffin  to  rise 
in  the  neck  of  the  flask  and  overflow  into  the  side  tube  and  bulb. 
After  sterilization  is  completed,  only  a  thin  layer  of  the  paraffin 
remains  on  the  top  of  the  bouillon,  and  during  heating  a  large  portion 
of  the  air  absorbed  by  the  bouillon  is  driven  out ;  its  re-absorption 
while  the  flask  is  cooling  is  prevented  by  the  thin  paraffin  film. 

2.  Before  inoculation  pierce  the  thin  paraffin  film  on  the  top  of 

*  A  normal  solution  of  caustic  potash  contains  as  many  grammes  to  the  litre  as 
the  number  of  its  molecular  weight — 56 '1  grammes  to  the  litre  of  water. 


68  PRACTICAL  BACTERIOLOGY 

the  bouillon,  and  when  the  inoculation  is  completed,  heat  the  side 
bulb  gently  to  melt  the  paraffin,  so  as  to  allow  it,  by  slightly  inclining 
the  flask,  to  pour  on  the  film  already  formed  above  the  bouillon. 
Upon  hardening,  this  additional  paraffin  forms  an  almost  air-tight 
layer,  and  when  the  flask  is  placed  in  the  incubator,  the  heat  causes 
the  paraffin  to  be  pressed  upwards  into  the  constricted  neck  of  the 
flask ;  this  closure  is  further  completed  by  the  gases  generated  in  the 
culture. 

§  CXXVIII.  THE  INCUBATOR. 

Certain  forms  of  bacteria  develop  at  a  higher  temperature  than 
others.  Pathogenic  or  disease-producing  organisms  grow  more 
luxuriantly  at  37'5°  C.  than  at  lower  temperatures,  whereas  the 
ordinary  saprophytic  forms  develop  almost  at  any  temperature 
between  18°  C.  and  37'5°  C.  For  the  cultivation  of  pathogenic 
bacteria  a  special  apparatus  is  used,  known  as  a  thermostat  or 
incubator,  made  throughout  of  copper,  with  double  walls,  the  space 
between  being  filled  with  water.  It  is  provided  with  a  closely- 
fitting  double  door  with  an  inner  door  of  glass,  enabling  the  cultures, 
etc.,  in  the  chamber  to  be  inspected  without  actually  opening  it.  The 
regulation  of  the  temperature  within  the  incubator  is  maintained  by 
an  automatic  regulator  of  sufficient  delicacy  to  prevent  a  fluctuation 
of  more  than  0'2°  C.  in  the  temperature  of  the  air  within  the  chamber 
of  the  apparatus.  A  Koch's  safety  burner  is  generally  used  for 
heating;  the  safety  attachment  automatically  turning  off  the  gas 
supply,  thus  preventing  accidents  should  the  flame  be  extinguished 
when  no  one  is  near. 

For  illustration  of  a  modern  incubator  and  various  attachments  in 
position,  see  Fig.  19,  p.  69. 

§  CXXIX.  SPECIAL  REACTIONS  PRODUCED  BY 
BACTERIA  DURING  THEIR  GROWTH. 

The  reactions  produced  by  many  species  of  bacteria  in  the  course 
of  their  development  in  culture  media  are  of  great  differential  value. 
These  changes  in  some  instances  are  so  great  that  they  can  be 
detected  by  simple  reagents,  whilst  in  others  they  are  so  slight  as  to 
require  the  most  delicate  test  for  their  demonstration.  Some  bacteria 
produce  at  one  period  of  their  life  an  alkaline  and  at  another  an  acid 
reaction.  This  is  seen  in  cultures  of  Loffler\s  bacillus  of  diphtheria. 

These  reaction  differences  are  best  observed  when  a  chemical 
substance,  which  does  not  interfere  with  the  development  of  the 


THE  INCUBATOR  69 

organism,  is  added  to  the  media.  In  milk  media,  to  which  litmus 
tincture  has  been  added  (see  §  85,  page  51),  organisms  pro- 
ducing alkalies  cause  the  blue  colour  to  be  intensified ;  others 
producing  acids  change  it  to  red ;  while  others  bring  about  neither  of 


FIG.  19.— Incubator. 

these  changes.     Tincture  of  litmus  can  also  be  added  to  gelatine  and 
agar-agar  for  the  same  purpose. 

In  ordinary  milk  cultures  coagula  also  appear,  due  to  acids  pro- 
auced  from  milk  sugar  by  bacterial  action  upon  the  casein  of  the 


70  PRACTICAL  BACTERIOLOGY 

milk ;    again    acids   may   be    produced    without    coagulation    being 
noticed. 

Rosolic  acid  in  alcoholic  solutions  (see  |  83,  page  51)  is  also 
added  to  culture  media  to  test  the  above  reactions. 

§  CXXX.  Fermentation^  or  the  production  of  gas  as  an  indication 
of  fermentation,  is  associated  with  the  growth  of  some  bacteria,  and 
is  best  seen  in  cultures  grown  in  media  containing  1  to  %  per  cent,  of 
grape  sugar.  Proceed  as  follows  :— 

1.  Liquefy  the  medium,  and  reduce  to  the  proper  temperature. 

2.  Place  a  small  quantity  of  a  pure  culture  of  the  organism  under 
investigation  in  the  liquid  medium  with  a  sterile  platinum  loop,  and 
distribute  it  equally. 

3.  Place  the  tube  in  ice-water  and  solidify  rapidly  in  a  vertical 
position. 

FIG.  21. 


FIG.  20. 


Smith's  Fermentation  Tube. 

Dunbar's  Fermentation  Tube. 

4.  When  solid,  place  in  the  incubator. 

5.  In   twenty-four   to   thirty-six   hours,  if  the    organism   causes 
fermentation  of  glucose,  the  medium  will  be  dotted  all   over  with 
small  cavities  containing  the  gas  formed. 

The  property  of  fermentation  with  production  of  gas  has  now 
assumed  such  an  important  role  as  a  means  of  differential  diagnosis 
that  not  only  the  amount  of  gas  or  gases  produced  by  an  organism 
under  consideration,  but  also  the  nature  and  quality,  are  determined. 
For  this  purpose  special  fermentation  tubes  are  necessary  (see  Figs. 
20  and  21.) 

It  is  a  tube  bent  at  an  acute  angle,  closed  at  one  end,  enlarged 
into  a  bulb  at  the  other,  and  attached  to  a  glass  foot,  so  that  it  may 
stand  upright.  The  tube  is  only  used  with  fluid  media  as  follows : — 


SPECIAL  REACTIONS  71 

1.  Pour  some  2  per  cent,  grape  sugar  bouillon,  made  just  before 
using,  into  the  bulb  of  the  tube  until  it  is  half  full. 

2.  Tilt  the  tube  until  the  closed  arm  is  nearly  horizontal,  so  that 
the  air  in  the  arm  may  escape  into  the  bulb  and  the  fluid  flow  into 
the  arm  to  take  its  place. 

3.  When  the  arm  is  completely  filled,  add  enough  of  the  liquid 
medium  to  cover  the  lowest  expanded  portion  of  the  bulb,  and  close 
the  opening  of  the  bulb  with  cotton  wadding. 

4.  Sterilize   the   tubes   on   three  consecutive    days  by   the  usual 
method. 

During  the  process  of  sterilization  the  tension  of  the  water  vapour 
in  the  arm  forces  most  of  the  fluid  into  the  bulb.  When  the  tube 
cools,  the  fluid  returns  again  into  the  arm,  except  in  a  small  space  at 
the  top,  occupied  by  air  originally  dissolved  in  the  liquid,  and  which 
was  driven  out  by  the  heat.  The  air  bubble  must  be  tilted  out  after 
each  sterilization,  and  finally  after  the  third  sterilization  the  arm  of 
the  tube  will  be  free  from  air. 

After  inoculation,  the  tubes  are  placed  in  the  incubator,  and  the 
amount  of  gas  collecting  in  the  closed  arm  noted  daily.  The  gas  is 
usually  found  to  consist  of  about  one  part  by  volume  of  carbonic  acid 
and  two  parts  by  volume  of  an  explosive  gas,  consisting  largely  of 
hydrogen. 

§  CXXXI.  METHOD  OF  DETERMINING  THE  NATURE 
AND  QUANTITATIVE  RELATIONS  OF  THESE 
GASES  ACCORDING  TO  SMITH. 

The  bulb  is  completely  filled  with  a  %  per  cent,  solution  of 
sodium  hydroxide  (NaOH),  and  closed  tightly  with  the  thumb. 
The  fluid  is  thoroughly  shaken  with  the  gas,  and  allowed  to  flow  to. 
and  fro  from  bulb  to  closed  branch  several  times  to  insure  intimate 
contact  of  the  CO2  with  the  alkali.  Lastly,  before  removing1  the 
thumb  all  the  gas  is  allowed  to  collect  in  the  closed  branch,  so  that  none 
may  escape  when  the  thumb  is  removed.  If  CO2  is  present,  a  partial 
vacuum  in  the  closed  branch  causes  the  fluid  to  rise  suddenly  when 
the  thumb  is  removed.  After  allowing  the  layer  of  foam  to  subside 
somewhat,  the  space  occupied  by  gas  is  again  measured,  and  the 
difference  between  this  amount  and  that  measured  before  shaking 
with  the  sodium  hydroxide  solution  gives  the  proportion  of  CO2 
absorbed. 

§  CXXXII.  Determine  the  explosive  character  of  the  residue  as 
follows : — 


72  PRACTICAL  BACTERIOLOGY 

Replace  the  cotton  plug,  and  allow  the  gas  in  the  closed  branch 
to  flow  into  the  bulb  and  mix  with  the  air  present  there.  The  plug 
is  removed,  and  a  lighted  match  inserted  into  the  mouth  of  the  bulb. 
The  intensity  of  the  explosion  varies  with  the  amount  of  air  present 
in  the  bulb. 


§  CXXXIII.  METHOD  OF   DETECTING  INDOL  IN 
CULTURES  OF  BACTERIA. 

1.  Cultivate  the  organism  from  twenty-four  to  forty-eight  hours  at 
37°  C.  in  Dunham's  Peptone  Solution   (see  §  82,  p.  51),  using  four 
tubes  kept  under  exactly  the  same  conditions. 

2.  Apply  the  test  as  follows  : — Take  two  tubes,  each  containing 
7  c.c.  of  the  peptone  solution,  but  '  not  inoculated.''     To  one  add  10 
drops  of  concentrated  sulphuric  acid,  to  the  other  1  c.c.  of  O'Ol  per 
cent,  solution   of  sodium  nitrite,  and  afterwards  10  drops  of  concen- 
trated   sulphuric   acid.     In    five    or    ten    minutes  if  no    rose  colour 
appears,  then  indol  is  absent. 

3.  To  two  '  inoculated '  tubes  add  10  drops  of  concentrated  sul- 
phuric acid,  and  in  five  or  ten  minutes,  if  no  rose  colour  appears,  add 
1  c.c.  of  the  sodium  nitrite  solution  ;  if  no  rose  colour  appears,  then  the 
'  indol  reaction  '  is  absent. 

4.  When  the  rose  colour  appears  with  the  addition  of  the  concen- 
trated sulphuric  acid  alone,  then  indol  has  been  formed,  and  likewise 
a  reducing'  body. 

5.  When  the  rose  colour  appears  only  with  the  addition  of  both 
the  concentrated  sulphuric  acid  and  the  nitrite  solution,  then  indol 
has  been  formed  during  the  growth  of  the  organism,  but  no  nitrites. 


§  CXXXIV.  TEST  FOR  NITRITES  IN  CULTURES. 

ILOSVAY'S  MODIFICATION  OF  GRIESS'S  METHOD. 
The  following  reagents  are  employed  :— 

A. 

Naphthylamine  ...  01  gramme. 

Aqua  distillata  .  .  .  20*0  c.c. 

Acetic  acid  (25  per  cent,  solution)        .          150'0  c.c. 
Dissolve  the  naphthylamine  in  20  c.c.  of  boiling  water,  allow  it  to 
cool,  and  mix  with  the  acetic  acid  solution. 


FILTRATION  OF  BACTERIA  73 

B. 

Sulphanilic  acid  0'5  gramme. 

Acetic  acid  (25  per  cent,  solution)       .          1 50'0  c.c. 
Mix  solutions  A  and  B  together;  the  resulting  mixture  should  be 
colourless.     The  solution  is  best  when  freshly  prepared,  but  if  kept  in 
a  close  stoppered  bottle  retains  its  power  for  some  time. 

Add  1  volume  of  the  above  solution  to  5  volumes  of  the  culture, 
when  if  nitrites  are  present  a  deep  red  colour  will  appear  in  a  few 
seconds  ;  if  no  nitrites  are  present  no  changes  take  place. 


§  CXXXV.  REICHEL'S  BACTERIA  FILTER. 

This  apparatus  consists  of  a  glass  flask  and  a  porcelain  filter  which 


FIG.  22. 


Fin.  23. 


Reichel's  Bacteria  Filter. 

fits  in  the  flask,  and  is  used  for  separating  bacterial  products  out  of 
culture  media  (see  Fig.  22). 


§  CXXXVL  METHOD  OF  USING  THE  FILTER. 

1.  Place   some  cotton  wadding  in  the  glass  tube  at  C,  and  also 
at  D. 

2.  The  porcelain  cylinder  B  is  placed  in  the  glass  flask,  and  both 
sterilized  in  the  hot-air  sterilizer,  a  circular  piece  of  asbestos  being 
placed  between  the  under  ring  of  the  porcelain  and  the  upper  surface 
of  the  neck  of  the  glass  flask. 

3.  When  the  apparatus  is  removed  from  the  hot-air  oven,  a  special 
perforated  rubber  cap  is  placed  over  the  top  of  the  filtering  cylinder 


74  PRACTICAL  BACTERIOLOGY 

and  the  glass  flask,  and  the  material  to  be  filtered  poured  into  the 
filter.  It  is  necessary  to  attach  a  piece  of  rubber  tubing  with  a  pinch- 
cock  at  D,  because  the  tube  C  is  attached  directly  to  the  tube  of  a 
suction-pump,  and  filtration  produced.  It  works  well  with  a  water 
force  of  1 J  atmospheres,  and  a  mercury  vacuum  meter  of  200  mm. 

§  CXXXVII.  INOCULATION  OF  ANIMALS. 

Inoculation  is  practised  on  animals  to  determine  if  an  organism  is 
pathogenic  or  disease-producing ;  and  if  so,  the  pathological  results  of 
its  growth  in  the  tissues  of  these  animals,  and  the  manner  in  which 
the  organism  gains  entrance  to  the  tissues  in  order  to  produce  these 
results.  The  animals  usually  employed  in  laboratories  for  inoculation 
purposes  are  white  mice,  grey  house  mice,  field  mice,  rats,  guinea-pigs, 
rabbits,  and  pigeons.  The  inoculations  are  made  subcutaneously, 
intravenously  into  the  great  serous  cavities,  or  into  the  anterior 
chamber  of  the  eye. 

§  CXXXVIII.  SUBCUTANEOUS  METHOD. 

1.  Remove  the  hair  or  feathers,  wash  the  skin  with  soap  and  water, 
and  sterilize  with  corrosive  sublimate  solution  1  to  1000  (see  §  66,  p.  44). 

2.  For  liquids  use  a  sterilized  hypodermic  syringe.     The  skin   is 
raised  with  a  pair  of  forceps,  and  the  point  of  the  hypodermic  needle 
inserted,  and  an  incision  made. 

3.  For  solid  material,  a  pocket  is  made  in  the  skin  as  follows  : — 
Take  up  the  skin  with  the  forceps,  make  a  small  incision  with  steril- 
ized scissors,  and  cut  a  pocket  under  the  skin.     Hold  the  pocket  open 
with  sterilized  forceps,  and  place  the  material  as  far  back  as  possible 
without  touching  the  edges  of  the  wound  with  the  platinum  loop  or 
KocKs  needle.     Earth  can  be  introduced  in  this  manner. 

4.  Pull  the    wound  together   and  allow  it    to  remain,  or  cover 
with    a    little    iodoform    collodion,    or   sear    with   a    hot   platinum 
needle.     During  the  operation  the  animal  must  be  held  perfectly  still. 
For  the  smaller  animals  many  forms  of  holders  are  made,  especially  for 
mice,  which  are  held  in  proper  position  for  inoculation  at  the  root  of 
the  tail.     Guinea-pigs,   rabbits,  and  pigeons   are  best   held   by  an 
assistant.     Pigeons  are  generally  inoculated  in  the  pectoral  muscles, 
mice  at  the  root  of  the  tail,  while  the  other  animals  are  generally 
inoculated  in  the  abdominal  wall,  either  to  the  right  or  left  of  the 
median  line. 


INOCULATION  OF  ANIMALS  75 


§  CXXXIX.  INTRAVENOUS  METHOD. 

In  the  rabbit  this  operation  is  generally  performed  in  one  of  the 
veins  of  the  ear,  and  the  most  suitable  vein  is  the  ramus  lateralis 
posterior  of  the  vena  auricularis  posterior,  a  very  fine,  delicate  vessel 
running  along  the  posterior  margin  of  the  ear ;  and  being  firmly  fixed 
in  the  dense  surrounding  tissue,  it  does  not  roll  about  when  you  are 
inserting  the  needle.  The  largest  branch  of  the  vena  auricularis 
posterior  is  the  central  branch,  or  ramus  anterior,  but  the  insertion  of 
a  needle  into  this  vessel  is  accomplished  with  difficulty. 

If  there  is  only  a  little  blood  in  the  ear,  pressure  at  the  root  of 
the  ear  will  cause  stasis  of  the  blood  and  distension  of  the  vessels, 
rendering  them  more  visible.  Another  method  of  rendering  a  vein 
more  prominent  is  to  lightly  press  or  gently  prick  with  the  point  of  a 
needle  the  skin  over  the  vessel  to  be  used.  The  injection  is  always 
made  from  the  dorsal  surface  of  the  ear.  Needles  employed  for  intra- 
venous injection  must  have  a  perfectly  flat  slanting  surface  free  from 
curvature. 

Care  must  be  taken  that  no  air  is  injected,  and  that  the  syringe 
and  needles  are  sterilized,  before  use,  in  the  steam  sterilizer  or  in 
boiling  water.  The  inoculated  animal  must  be  kept  under  close 
observation  for  an  hour  after  the  operation. 

The  syringes  generally  used  are  Koch's,  Strohschein's,  and  Over- 
lack's  ;  but  the  latter  is  preferred,  as  the  dose  can  be  controlled  with 
accuracy  owing  to  the  amount  of  air  between  the  fluid  and  the  piston 
being  at  a  minimum. 

§  CXL.  INOCULATION  INTO  THE  LYMPHATIC 
CIRCULATION. 

Fluid  cultures  or  suspensions  of  bacteria  can  be  injected  into  the 
lymphatics  by  way  of  the  testicles,  by  plunging  the  point  of  the 
needle  into  the  substance  of  the  testicle  and  injecting  the  desired 
amount  of  fluid. 

§  CXLI.  INOCULATION  INTO  THE  GREAT  SEROUS 

CAVITIES. 

To  inject  fluid  into  the  peritoneum,  make  a  small  nick  through 
the  skin,  and  plunge  the  needle  direct  into  the  peritoneal  cavity. 
There  is  not  much  danger  of  wounding  the  intestines  and  other 


76  PRACTICAL  BACTERIOLOGY 

viscera  when  the  inoculation  is  made  close  to  the  median  line,  half-way 
between  the  sternum  and  the  symphysis  pubis,  and  a  curved  needle 
used,  with  an  opening  some  distance  from  the  point  on  the  convex  side. 
When  solid  substances,  bits  of  tissue,  etc.,  are  to  be  introduced, 
the  operation  must  be  conducted  on  the  lines  of  a  laparotomy,  as 
follows : — 

•1.  Shave  the  hair  from  a  small  area  over  the  median  line,  wash 
the  skin  with  soap  and  water,  apply  fresh  water,  rub  with 
alcohol,  and  finally  sterilize  with  corrosive  sublimate 
solution  1  to  1000  (see  §  66,  p.  44). 

%.  Make  a  longitudinal  incision  about  2  c.  long,  close  to  the 
median  line,  through  the  skin  and  down  to  the  fascia. 

3.  Two    subcutaneous    sutures,   as    employed    by   Hals  ted,   are 

introduced  transversely  to  the  line  of  incision  about  1  c. 
apart,  and  their  ends  left  loose.  These  sutures  do  not 
pass  through  the  skin  proper,  but  are  introduced  into  the 
subcutaneous  tissues,  passing  into  the  abdominal  cavity 
and  out  again,  entering  at  one  side  of  the  line  of  incision 
and  leaving  at  the  other. 

4.  The  remaining  tissues  are  now  cut  through,  and  the  bit  of 

tissue   deposited   in   the  peritoneal  cavity  (under   sterile 

precautions),   the   edges  of    the    incision    closed   tightly 

and  evenly  by  drawing  and   tying  the  sutures,  and  the 

line  of  incision  dressed  with  iodoform  collodion. 

All  instruments,  sutures,  ligatures,  etc.,  used  in  the  operation  are 

previously  carefully  sterilized,  either  in  the  steam  sterilizer,  or  boiled 

in  a  2  per  cent,  solution  of  sodium  carbonate  for  ten  minutes,  and  the 

operator's   hands  cleansed  with   disinfecting   solution  *(§  66).      The 

material  placed  in  the  abdominal  cavity  must  also  be  handled  with 

sterilized  instruments. 


§  CXLIL  INOCULATION  INTO  THE  PLEURAL  CAVITY. 

This  is  very  seldom  practised,  as  it  is  very  difficult  to  enter  the 
pleural  cavity  without  injuring  the  lung. 


§  CXLIII.   INOCULATION  INTO  THE  ANTERIOR 
CHAMBER  OF  THE  EYE. 

A   puncture   is   made  through  the  cornea  just   in   front  of  its 
junction    with    the   sclerotic,   the    knife   passing    into   the   anterior 


DIAGNOSIS  OF  RABIES  77 

chamber  in  a  plane  parallel  to  the  plane  of  the  iris,  when  the  aqueous 
humour  flows  out.  A  few  drops  of  a  2  per  cent,  solution  of  cocaine 
are  placed  in  the  eye  previous  to  operating.  Deposit  the  bit  of  tissue 
with  fine  sterilized  forceps  or  a  platinum  loop  through  the  opening 
upon  the  iris,  where  it  remains,  and  its  pathogenic  properties  upon 
the  iris  can  be  conveniently  studied.  The  application  of  this  mode 
of  inoculation  is  very  limited.  Cohnheim  employed  this  method  in 
demonstrating  the  infectious  nature  of  tuberculous  tissues,  tubercular 
iritis  being  a  constant  symptom  when  tubercular  matter  was  intro- 
duced into  the  anterior  chamber  of  the  rabbit's  eye. 

§  CXLIV.  METHOD  OF  INOCULATING  RABBITS  FOR 
THE  DIAGNOSIS  OF  RABIES. 

1.  Remove  the  brain  of  the  suspected  animal,  with  antiseptic  pre- 
cautions, as  soon  as  possible  after  death. 

2.  Place  a  small  piece  of  the  brain  or  spinal  cord  in  a  mortar,  and 
thoroughly  grind  with  a  few  c.c.  of  sterile  water  or  bouillon. 

3.  The   operator    must    disinfect    the    hands    and    sterilize    all 
instruments. 

4.  Anaesthetize  the  rabbit  with  ether,  clip  the  hair  from  the  head 
between  the  eye  and  ear,  wash  the  skin,  and  disinfect  with  ordinary 
sublimate  solution  (§  66,  p.  44). 

5.  A  longitudinal  incision  is  made  through  the  skin  and  sub- 
cutaneous tissue  in  the  median  line,  while  a  crucial  incision  is  made 
through  the  periosteum  on  one  side  of  the  median  line,  thus  avoiding 
haemorrhage  from  the  longitudinal  sinus,  and  the  four  parts  of  the 
periosteum  reflected  or  pushed  back.     Cut  out  a  piece  of  bone  about 
^  of  an  inch  in  diameter  with  a  trephine  and  expose  the  dura  mater. 

6.  Inject  a  drop  or  more  of  rabid  material  beneath  the  dura  mater 
with  a  hypodermic  syringe,  replace  the  periosteum,  suture  the  skin, 
disinfect,  dry,  and  apply  some  iodoform  collodion.     Sometimes  a  piece 
of  the  suspected  tissue  may  be  introduced  directly  under  the  dura 
mater.     The  inoculation  wound  heals  rapidly.     Rabid  symptoms  may 
appear  in  fifteen  to  thirty  days,  sometimes  earlier  than  fifteen  days, 
and  again  from  one  to  three  months  may  intervene. 

§  CXLV.  OBSERVATION  OF  ANIMALS  AFTER 
INOCULATION. 

Inoculated  animals  must  be  kept  under  constant  observation,  and 
the  following  conditions  noted  : — 


78  PRACTICAL  BACTERIOLOGY 

1.  Temperature. 

2.  Loss  of  weight. 

3.  Peculiar  position  in  cage. 

4.  Loss  of  appetite. 

5.  Condition  of  the  coat  or  hair. 

6.  The  condition  of  the  secretions  from   the  air  passages,  con- 
junctiva, kidneys,  diarrhoea  or  haemorrhage  from  the  bowels. 

7.  The  condition  of  the  seat  of  inoculation.    When  an  animal  dies 
in  two  to  four  days  after  inoculation,  evidence  of  either  acute  or  toxic 
processes  will  be  found. 

When  the  inoculation  produces  chronic  conditions  the  animal 
may  be  under  supervision  for  weeks,  and  must  be  weighed  daily  at 
the  same  hour,  the  temperature  taken  at  the  rectum,  the  thermometer 
being  pushed  past  the  sphincter. 

Too  much  stress  must  not  be  laid  upon  moderate  and  sudden 
daily  fluctuations  in  either  temperature  or  weight,  as  normal  animals 
when  confined  in  cages  and  fed  regularly  present  striking  temporary 
gains  and  losses  in  weight,  and  unexplainable  rises  and  falls  of 
temperature,  often  as  much  as  a  degree  from  one  day  to  another. 

§  CXLVI.  POST-MORTEM  EXAMINATION  OF  ANIMALS. 

Perform  the  autopsy  as  soon  as  possible  after  death.  When  delay 
cannot  be  avoided,  place  the  animal  in  the  ice-chest  until  such  time  as 
is  convenient. 

A. 

1.  Sterilize  a  suitable  post-mortem  board  with  corrosive  sublimate 
solution  1  to  1000,  place  the  animal  belly  upwards,  and  tack  the  four 
legs  fast  to  the  board. 

2.  Wash  the  surface  of  the  thorax  and  abdomen  with  corrosive 
sublimate  solution,  make  an  incision  through  the  skin  at  the  pubis, 
introducing  one  blade  of  the  scissors,  and  extend  the  incision  as  far  as 
the  chin. 

3.  Carefully  dissect  the  skin  away  from   the  abdomen,  thorax, 
axillary,  inguinal,  and  cervical  regions,  and  fore  and  hind  legs,  and 
pin  it  to  the  board  as  far  as  possible  from  the  thorax  and  abdomen. 
It  is  from  the  skin  that  the  chances  of  contamination  are  greatest. 

B. 

All  incisions  from  now  on  are  made  with  sterilized  instruments. 
1.  Take  an  ordinary  potato-knife,  heat  it  quite  hot,  and  place  it 


POST-MORTEM  EXAMINATION  OF  ANIMALS  79 

in  the  abdomen  in  the  region  of  the  linea  alba  until  the  fascia  begins 
to  burn,  when  the  knife  is  then  held  transverse  to  this  line  over  the 
centre  of  the  abdomen,  making  two  sterilized  tracks  through  which  the 
abdomen  may  be  opened  by  crucial  incisions ;  two  burned  lines  are 
also  made  along  the  sides  of  the  thorax. 

2.  Make  a  central  longitudinal  incision  from  the  sternum  to  the 
genitalia  with  heated  scissors,  the  abdominal  wall  being  held  up  with 
sterilized  forceps,  or  a  hook,  to  prevent  the  internal  viscera  being 
injured.     A  transverse  incision  is  made  in  a  similar  manner. 

3.  Cut  through  the  ribs  with  strong  sterilized  scissors  along  the 
sterilized  tracks  in  the  sides  of  the  thorax,  when  the  whole  anterior 
wall  of  the  thorax  is  easily  lifted  and  entirely  removed  by  severing 
the  diaphragm  connections. 

4.  When  the  thoracic  and  abdominal  cavities  are  fully  exposed  a 
careful  examination  of  the  organs  and  surroundings  is  made  without 
disturbing  them. 

5.  Plate,  Petri-dish,  or  roll  cultures  are  prepared  from  the  blood, 
liver,  spleen,  kidneys,  and  any  exudates  present. 

The  method  is  as  follows : — 
(1.)  Heat  a  scalpel  and  scorch  a  small  surface  of  the  organ  from 

which  the  cultures  are  to  be  made. 

(2.)  Heat  the  scalpel  again  and  penetrate  the  capsule  of  the  organ 
with  the  point,  and  through  the  opening  insert  a  stout 
sterilized  platinum  loop,  push  it  into  the  tissues,  twist 
around,  and  obtain  enough  material  from  the  centre  of 
the  organ  to  make  the  culture. 

In  making  cultures  from  resisting  tissues  NuttalPs  platinum  spear 
can  be  used  to  advantage.  It  is  a  piece  of  heavy  platinum  with  a 
spear  head  at  one  end  perforated  with  a  small  hole,  the  other  end 
being  attached  to  either  a  metal  or  glass  holder.  When  heated  it 
can  be  readily  thrust  into  the  densest  of  the  soft  tissues,  and  when 
withdrawn  after  twisting,  sufficient  material  will  be  found  in  the  eye 
of  the  spear  head  for  examination  or  the  institution  of  cultures. 

Cultures  from  blood  are  usually  made  from  one  of  the  heart 
cavities,  the  surface  being  seared  with  a  hot  knife  before  opening. 
As  soon  as  the  culture  material  is  obtained,  cover-glass  specimens  are 
prepared  from  each  organ  and  existing  exudates. 

Small  pieces  of  each  organ  are  also  preserved  for  future  examina- 
tion.   (For  method,  see  Examination  of  Organs  and  Tissues,  §  31,  p.  30.) 
When  the  autopsy  is  finished  the  remainder  of  the  animal  should 
be  burned  and  the  instruments  used  sterilized  by  the  process  given 
under  Sterilization  (see  p.  12).     Wash  the  post-mortem  board  with 


80  PRACTICAL  BACTERIOLOGY 

sublimate  solution  1  to  1000,  and  sterilize  in  the  steam  sterilizer 
The  cover-glasses  and  other  material  likely  to  contain  infectious 
material  must  also  be  further  sterilized  when  of  no  further  use. 


METHODS    OF    EXAMINING    AIR,   WATER, 
AND    EARTH. 

§  CXLVII.  AIR— ORDINARY  METHOD. 

1.  Liquefy  10  c.c.  of  sterile  gelatine,  pour  on  a  sterile  plate  or  in 
a  Petri-dish,  and  set  aside  to  cool. 

2.  Remove  the  cover  of  the  dish  or  plate,  leaving  the  gelatine 
exposed  for  one  hour. 

3.  Replace  the  cover,  set  aside,  and  examine  any  future  develop- 
ment. 

Agar  can  be  used  in  place  of  gelatine.     This  method  yields  only 
qualitative  results. 

§  CXLVIII.  AIR— HESSE'S  METHOD. 

1.  A  sterilized  glass  cylinder,  70  c.  long  and  3  to  5  c.  in  diameter, 
containing  50  c.c.  of  sterile  gelatine  adhering  to  the  inner  surfaces, 
prepared  according  to  the  Roll  Culture  process  (see  §  114,  p.  61),  with 
one  end  of  the  cylinder  closed  by  two  elastic  caps,  the  inner  one 
having  a  central  orifice,  and  the  other  with  a  cork  and  glass  tube 
plugged  with  cotton  wool. 

2.  The  prepared  apparatus  is  fitted  on  a  tripod.    The  cotton  wool 
plug  is  removed,  and   the  small  tube  connected   with  an  aspirator 
capable  of  drawing  air  through  the  cylinder  at  a  velocity  of  half  a 
litre  per  minute. 

3.  Before  the  aspirator  is  finally  adjusted,  the  outer  rubber  cap  at 
the  opposite  end  of  the  cylinder  is  removed,  when  any  germs  passing 
into  the  cylinder  adhere  to  the  gelatine. 

About  20  litres  of  air  is  the  limit  tested  by  this  method.     (For 
illustration  of  apparatus,  see  Fig.  24,  p.  81.) 

§   CXLIX.  AIR— PETBI'S  METHOD. 

By  this  method  a  greater  quantity  of  air  can  be  tested  than  by 
Hesse's  method. 

1.  Prepare  a  glass  tube  9  c.  long  and  1*5  c.  in  diameter,  containing 


METHODS  OF  EXAMINING  WATER 


81 


two    filters  of  fine  sand   separated   from   each   other  by  wire-gauze 
netting. 

2.  After  the  tube  is   sterilized  and  plugged,  it  is  placed   in  a 
horizontal  position,  and  the  proximal  end  of  the  tube  attached  to  an 
air-pump  (see  Fig.  25,  page  82),  capable  of  drawing  from  5  to  10 
litres  of  air  per  minute,  and  the  plug  at  the  distal  end  of  the  tube 
removed. 

3.  The  air-pump  is  worked  until  the  gauge  registers  100  litres. 

4.  The  sand  filter  at  the  distal  end  of  the  tube  is  removed  and 
mixed  with  10  c.c.  of  sterilized  liquefied  gelatine,  which  is  poured 
into  a  sterile  Petri-dish.     The  sand  filter  at  the  proximal  end  of  the 


FIG.  24.— Hesse's  Apparatus  for  Examining  Air. 

tube  is  used  as  a  control,  and  must  remain  germ  free  when  mixed  with 
nutrient  gelatine,  and  poured  into  a  Petri-dish. 

Powdered  glass  can  be  substituted  for  sand,  and  is  more  satis- 
factory, as  any  developing  colonies  can  be  readily  observed. 

§  CL.  METHODS  OF  EXAMINING  WATER. 

Samples  of  water  can  be  procured  in  sterilized  Erlenmeyer  flasks 
closed  with  cotton-wool  plugs.  They  should  be  examined  immedi- 
ately, if  possible,  and  never  later  than  two  hours  after  the  sample  is 

F 


82  PRACTICAL  BACTERIOLOGY 

obtained.  When  the  examination  must  be  delayed,  then  place  the 
sample  in  the  ice-box.  Before  testing  a  sample,  shake  the  flask,  as  an 
equal  distribution  of  the  germs  is  necessary  for  an  average  result. 
Samples  collected  from  a  tap  should  not  be  drawn  until  the  water  has 
been  flowing  from  fifteen  to  twenty  minutes  in  full  stream.  When 
obtained  from  a  stream  or  spring,  take  the  sample  about  one  foot 
beneath  the  surface. 

Water  analyses  should  always  be  made  on  the  spot,  when  possible, 
as  during  transportation,  unless  packed  in  ice,  a  multiplication  of  the 


FIG.  25.— Air-pump  for  use  in  Petri's  Method. 

organisms  in  the  sample  takes  place.    The  following  are  the  necessary 

articles  for  a  Transportation  Case  for  Analyses  on  the  Spot : — 

4  sterilized  Erlenmeyer  flasks  to  obtain  the  samples  ;  1  thermo- 
meter ;  1  spirit  lamp  ;  12  sterilized  Petri-dishes  in  special 
box  (see  page  12) ;  12  tubes  of  sterile  nutrient  gelatine  ; 
15  sterilized  water  pipettes  in  three  cases  ;  1  folding  tripod  ; 
1  notebook  ;  1  pencil  for  writing  on  "glass,  etc. ;  and  1  towel. 
The  gelatine  is  melted,  the  inoculations  made  immediately,  and 

poured  into  the  Petri-dishes,  allowed  to  solidify,  and  transported  to 

the  laboratory. 


METHODS  OF  EXAMINING  WATER  83 


§  CLI.  QUALITATIVE  METHOD. 

Transfer  with  a  sterilized  capillary  pipette  1  c.c.,  J  c.c.,  or  J  c.c. 
of  the  water  to  be  examined  into  a  tube  containing  10  c.c.  of  liquefied 
sterilized  nutrient  gelatine.  Mix,  and  pour  the  contents  of  the  tube 
on  a  sterile  glass  plate,  and  proceed  as  at  No.  3  process,  '  Quantitative 
Plate  Culture  Method?  §  118,  p.  64.  (The  bottoms  of  Petri-dishes 
being  uneven,  plates  are  more  reliable  for  water  examination.) 

§  CLII.  KOCH'S  METHOD  FOR  DEMONSTRATING  THE 
VIBRIO  CHOLERA  ASIATICS  IN  WATER. 

1.  Take  100  c.c.  of  the  suspected  water,  and  mix  with  5  c.c.  of 
sterile  20  per  cent,  peptone  chloride  of  sodium  solution  (§  81,  p.  50), 
make  alkaline,  and  place  in  the  incubator  at  37°  C. 

2.  When  Cholera  bacilli  are  present  they  develop,  and  are  found 
in  ten  or  twelve  hours  on  the  surface  of  the  fluid,  and  can  be  further 
investigated  and  identified. 

§  CLIII.  ELSNER'S  METHOD  FOR  BACILLUS  TYPHI 
ABDOMINALIS. 

Take  some  potato  gelatine  (see  §  79,  p.  50),  and  shortly  before  use 
add  1  per  cent,  of  iodide  of  potash ;  after  adding  the  suspected  water, 
prepare  plate  cultures  according  to  the  ordinary  method  (§  108,  p.  58). 
This  method  is  for  the  Bacillus  typhi  abdominalis,  and  the  Bacterium 
coli  com  munis,  which  grows  the  stronger  of  the  two,  and  in  forty-eight 
hours  appears  as  dull  brown  colonies,  while  the  Bacillus  typhi  abdo- 
minalis appears  as  bright  watery  drops.  This  method,  however,  is  not 
absolutely  reliable. 

§  CLIV.    QUICK  METHOD  OF  DEMONSTRATING  THE 
PRESENCE  OF  PATHOGENIC  GERMS  IN  WATER. 

1.  Mix  100  c.c.  of  the  suspected  water  with  5  c.c.  of  Koch's  20  per 
cent,  peptone  chloride  of  sodium  solution  (see  §  81,  p.  50),  and  place 
in  the  incubator  twenty-four  hours  at  37°  C. 

2.  Inoculate  a  guinea-pig  with  1  c.c.   of  the  mixture  intraperi- 
toneally,  and  if  pathogenic  organisms  are  present,  the  animal  dies, 
and  the  organs,  exudates,  blood,  etc.,  can  be  further  examined. 

3.  If  the  water  is  pure,  the  guinea-pig  remains  alive. 


84  PRACTICAL  BACTERIOLOGY 

§  CLV.  SMITH'S  METHOD  OF  ISOLATING  CERTAIN 
ORGANISMS  FROM  A  WATER  SUPPLY,  i.e.,  INTES- 
TINAL BACTERIA. 

1.  Add  1, 2,  or  3  drops  of  the  suspicious  water  to  some  fermentation 
tubes  containing  2  per  cent,  grape  sugar  bouillon. 

2.  Place  at  37°  C.,  and  if  at  the  end  of  thirty-six  or  forty-eight 
hours  gas  accumulates  in  the  tube,  then  intestinal  bacteria  are  present, 
as  ordinary  water  bacteria  do  not  flourish  in  this  medium. 

3.  Isolate  the  gas-forming  organisms  by  the  ordinary  plate  culture 
method  (see  §  108,  p.  58)  for  further  identification. 

When  the  number  of  colonies  developed  on  a  plate  are  too 
numerous  to  be  counted  with  the  Wolff hiigePs  apparatus  (see  Fig.  13, 
p.  63),  the  plate  is  examined  with  a  low  power  and  Ehrlich's  eye-piece 
diaphragm. 

§  CLVI.  METHODS  OF  EXAMINING  EARTH. 

FRAENKEL'S  METHOD. 

1.  Take  an  earth  borer  made  according  to  FraenkeFs  model  (see 
Fig.  26,  A  open,  B  closed)  when  examining  earth  from  the  deeper 
strata.  For  superficial  samples  a  borer  is  not  necessary. 


Open 


Fraenkel's  Earth  Borer. 


2.  Measure  the  desired  quantity  of  earth  in  a  platinum  spoon, 
mix  with  10  c.c.  of  sterilized  nutrient  gelatine,  and  proceed  according 
to  roll  culture  process  (see  §  114,  p.  61). 

The  most  important  organisms  found  in  soil  are  Bacillus  tetani, 
Bacillus  of  symptomatic  anthrax,  Bacillus  oedematis  maligni.  These 
organisms  are  anaerobic,  and  require  special  conditions  for  their 
development  (see  Cultivation  of  Anaerobic  Bacteria,  §  119,  p.  64). 

The  nitrifying  bacteria  are  also  found  in  the  most  superficial 
layers  of  the  soil.  The  best  example  of  the  activity  of  these 
organisms  is  the  production  of  nitrates  from  the  ammonia  of  the 


METHODS  OF  EXAMINING  EARTH  85 

fsecal  evacuations  of  sea  fowls  in  the  saltpetre  beds  of  Chili  and  Peru. 
It  is  largely  by  means  of  these  organisms  that  growing  vegetation 
obtains  the  nitrogen  necessary  for  the  nutrition  of  its  tissues.  Special 
cultivation  media  are  necessary  for  the  growth  of  these  organisms. 

WINOGRADSKY'S  METHOD. 

For  preparation  of  the  special  media   used,  see  Preparation  of 
Media  (§  105,  p.  57). 

1.  Inoculate  one  of  the  flasks  with  a  little  of  the  soil  to  be  investi- 
gated. 

2.  In  four  or  five  days  withdraw  with  a  sterile  pipette  some  of 
the  solution  from  the  above  flask  from  over  the  surface  of  the  layer  of 
magnesia  carbonate,  and  transfer  to  a  second  flask  No.  2. 

3.  Change  again  some  material  from   flask  No.  £  in  four  or  five 
days  to  a  third  flask,  continue  the  changing  process  until  ultimately 
the  nitrifying  organisms  flourish  as  almost  transparent  films  attached 
to  the  granulated  magnesium  carbonate  at  the  bottom  of  the  flask. 
The  organism  is  known  as  the  Nitromonas. 

§  CLVIL  POINTS  TO  BE  OBSERVED  IN  DESCRIBING  AN 
ORGANISM— (ABBOTT). 

1.  Its  source — as  air,  water,  or  soil.     If  found  in  the  animal  body, 
is  it  normally  present,  or  only  in  pathological  conditions  ? 

2.  Its  form,  size,  mode  of  development,  occurrence  of  involution 
forms,  or  other  variations  in   morphology.      Grouping,  as  in  pairs, 
chains,    clumps,    zoogloea;    presence    of   capsules;   development   and 
germination  of  spores  ;  arrangement  of  flagella. 

3.  Staining   peculiarities — especially    its    reactions    with    Gram's 
(or  Weigert's  fibrin)  stain,  and  peculiar  or  irregular  modes  of  staining. 

4.  Motility — to  be  determined   on  very  fresh   cultures   and   on 
cultures  in  different  media. 

5.  Its  relation  to  oxygen — Is  it  aerobic,  anaerobic,  or  facultative  ? 
Does  it  develop  in  other  gases,  as  carbonic  acid,  hydrogen,  etc. 

6.  Both    the    macroscopic   and    microscopic    appearance    of    its 
colonies  on  nutrient  gelatine  and  on  nutrient  agar-agar. 

7.  The  appearance  of  its  growth  in  stab  and  slant  cultures  on 
gelatine,  agar-agar,  blood  serum,  and  on  potato. 

8.  The  character  of  its  growth  in  fluid  media,  as  in  bouillon, 
milk,  litmus   milk,  rosolic  acid,  peptone  solution,  and  in   bouillon 
containing  glucose. 

9.  Does  it  grow  best  in  acid,  alkaline,  or  neutral  media  ? 


86  PRACTICAL  BACTERIOLOGY 

10.  Is  the  normal  reaction  of  the  medium  altered  by  its  growth  ? 
Is  its  growth  accompanied  by  the  production  of  indol  ?  Is  the  indol 
associated  with  the  coincident  production  of  nitrites  ? 

11.  Is    its    growth   accompanied   by   the   production    of  gas,  as 
evidenced  by  the  appearance  of  gas  bubbles  in  the  media,  both  in 
media  containing  fermentable  sugars  and   those  from  which   these 
bodies   are   absent  ?       When   cultivated   in   sugar   bouillon   in   the 
fermentation  tube,  what  production  of  gas  is  evolved  under  known 
conditions  ?     How  much  of  this  gas  is  carbonic  acid  and  how  much  is 
explosive  ? 

12.  At  what  temperature  does  it  thrive  best,  and  the  lowest  and 
highest  temperature  at  which  it  will  develop  ?     What  is  its  thermal 
death-point,  both  by  steam  and  dry-air  methods  of  determining  this 
point  ? 

13.  What  is  its  behaviour  when  exposed  to  chemical  disinfectants 
and  antiseptics  ?      Does   it  withstand   drying   and   other   injurious 
influences,  both  in  the  vegetative  and  spore  stages  ?     The  germicidal 
value  of  the  blood  serum  of  different   animals  may  also   be   tried 
upon  it. 

14.  Its  pathogenic  powers — modes  of  inoculation  by  which  these 
are  demonstrated  ;  quantity  of  material  used  in  inoculation  ;  duration 
of  the  disease  and  its  symptoms  ;  lesions  produced,  and  distribution 
of    the    bacteria    in   the   inoculated    animal ;    which    animals    are 
susceptible  and  which  immune,  and  the  character  of  its  pathogenic 
activities  ?     Variations  in  virulence,  and  the  probable  cause  to  which 
they  are  due.     Can  they  be  produced  artificially  and  at  will  ? 

15.  The  detection  of  specific,  toxic,  and  immunizing  products  of 
growth. 

16.  Its  behaviour  when  exposed  to  the  influence  of  blood  serum  of 
animals  immunized  from  it;    also  its  behaviour  when  mixed  with 
serum  from  an  animal  in  the  height  of  infection  by  it.     Are  the 
relations  between  the  organism  and  the  serum  constant  and  specific  ? 


PART    III. 

SPECIAL    BACTERIOLOGY. 

BACTERIA    FOUND    IN    INFLAMMATION    AND 
SUPPURATION. 

NEARLY  all  bacteria  produce  under  certain  conditions  inflammation 
and  suppuration.  These  changes  can  also  be  produced  by  chemical 
substances — acetic  acid,  ammonia,  oil  of  turpentine,  etc.,  but  above 
all  by  the  products  (ptomaines,  proteine,  etc.)  separated  from  bacteria. 
The  bacteria  mostly  associated  with  ordinary  inflammatory  and 
suppurative  processes  are  : — 

1.  The  pyogenic  cocci,  staphylococci,  streptococci,  pneumococci, 

etc. 

2.  The  Bacillus  coli  communis  and  allied  members  of  that  group. 

3.  The  rarely  present  Friedlander's  pneumobacillus. 

4.  Bacillus  pyocyaneus. 

STAPHYLOCOCCUS  PYOGENES  AUREUS. 

Microscopical  Appearances. — Micrococci  from  0-7  to  1-2  p  in 
diameter,  usually  arranged  together  like  bunches  of  grapes.  (See  Photo- 
micrograph, Plate  IV.,  Fig.  22.) 

Motility. — Non-motile. 

Staining  Reactions. — They  are  easily  stained  with  all  the  basic 
anilin  dyes,  and  by  the  Gram  and  Cladius  methods. 

Biological  Characters.  —  Facultative  anaerobe,  producing  the 
yellow  pigment  only  in  the  presence  of  oxygen.  The  minimum 
temperature  for  their  growth  is  6°  C.,  maximum  44°  C.,  optimum  34°  to 
38°  C. 

On  Gelatine  Plates.  —  Examined  under  a  low  power  they  form  in 
the  beginning  round,  coarsely  granular  colonies,  of  a  greyish -white  colour, 


88  SPECIAL  BACTERIOLOGY 

with  sharply  defined  borders ;  later  the  colonies  assume  a  yellow  colour 
and  liquefy  the  gelatine  quickly. 

In  Gelatine  Stab  Cultures.  —  The  development  takes  place  along 
the  whole  length  of  the  puncture,  with  accompanying  liquefaction  of 
the  medium,  the  growth  resembling  a  stocking. 

On  Agar  Stroke  Cultures.  —  It  forms  a  moist,  shiny,  gold-coloured 
elevated  growth ;  a  similar  growth  occurring  on  potatoes. 

Bouillon  becomes  densely  clouded  with  a  yellow  sediment. 

Milk  is  coagulated. 

In  milk  and  bouillon  lactic  acid  is  chiefly  formed. 

Vitality. — Cultures  remain  alive  for  one  year,  and  are  killed  in  a 
short  time  in  the  steam  sterilizer.  On  silk  threads  saturated  with 
staphylococci  pus,  and  dried,  the  cocci  are  killed  by  2  to  3  per  cent, 
carbolic  acid  in  five  minutes. 

Pathogenesis. — Cutaneous  inoculation  is  negative,  but  subcutaneous 
injection  causes  a  local  abscess  in  mice,  guinea-pigs,  and  rabbits ;  and 
intravenous  injection  in  rabbits  sometimes  causes  pyaemia. 

Staphylococcus  pyogenes  albus. — This  coccus  is  identical  with 
the  aureus,  excepting  the  absence  of  the  golden  pigment. 

Staphylococcus  pyogenes  citreus. — This  coccus  produces  a  citron- 
yellow  pigment,  but  in  other  respects  resembles  the  aureus. 

Staphylococcus  cereus  albus  and  cereus  flavus  are  seldom 
found,  but  are  characterised  by  not  liquefying  gelatine  media ;  the  one 
exhibits  a  waxy  white  pigment,  the  other  a  waxy  yellow  pigment. 

DIFFERENTIAL    TABLE. 

A.   The  Gelatine  is  liquefied. 

1.  Staphylococcus  pyogenes  aureus. 

2.  „  „  citreus. 

3.  „  „  rosaceus. 

4.  „  „  albus. 

B.   The  Gelatine  is  not  liquefied. 

1.  Staphylococcus  cereus  flavus. 

2.  „  „        griseus. 


The  Staphylococcus  pyogenes  aureus  is  found  in  furunculus, 
carbuncles,  acute  abscesses,  circumscribed  phlegmons  of  the  skin, 
impetigo,  sycosis,  blepharo-adenitis,  conjunctivitis  phlyctenulosa, 
acute  infectious  osteomyelitis,  suppuration  of  lymph-glands,  empyema, 
articular  and  bursal  suppuration,  tonsillar  abscesses,  mammary 


STREPTOCOCCUS  PYOGENES  89 

abscesses,  suppuration  of  the  parotid,  idiopathic  cerebro  -  spinal 
meningitis,  strumitis,  and  suppurative  peripleuritis.  The  albus  is 
also  often  present  in  these  infections — mixed  infection  being  frequent. 


STREPTOCOCCUS  PYOGENES. 

Microscopical  Appearances. — Micrococci     from  0*3  to    1    /x  in 
diameter,  arranged  in  more  or  less  long  chains. 
Motility. — Non-motile. 

Staining  Reactions. — Easily  stained  with  all  the  basic  anilin  dyes, 
and  by  the  Gram  and  Cladius  methods. 

Biological  Characters. — Facultative  anaerobe,  the  optimum  tem- 
perature for  its  growth  being  30°  to  37°  C.  ;  it  also  grows  at  room  tem- 
perature. 

On  Gelatine  Plates  it  develops  in  the  form  of  small  white  granular 
colonies  which  do  not  liquefy  the  gelatine.  Under  a  high  power  chains 
can  be  observed  projecting  from  the  sides  of  the  discs. 

In  Gelatine  Stab  Cultures  the  growth  is  not  confluent,  but  individual 
colonies  are  arranged  next  each  other  along  the  track  of  the  needle.  A 
similar  growth  occurs  in  agar  stab  cultures. 

Bouillon,  which  is  an  excellent  medium  for  streptococci,  is  not  clouded 
throughout,  but  a  flaky,  creamy  deposit  is  formed. 

On  Potatoes  the  growth  is  extremely  scanty. 

Milk  is  coagulated. 

Vitality. — In  cultures  the  streptococcus  dies  much  sooner  than  the 
staphylococcus,  only  living  about  four  months.  In  order  to  keep 
streptococci  cultures  virulent,  Petruschky  uses  gelatine  cultures,  and 
renews  them  every  five  days,  and  keeps  them  in  the  ice-chest. 

Pathogenesis. — Material  containing  streptococci,  when  rubbed  on 
an  abrased  surface  on  a  rabbit's  ear,  causes  an  erysipelatous  inflamma- 
tion ;  when  introduced  into  mice  or  rabbits  a  septicaemia  results, 
with  or  without  a  local  abscess.  Intravenous  injection  causes 
septicaemia.  Many  suppurative  processes  spread  by  means  of  the 
lymphatics,  causing  lymphangitis  and  lymphadenitis.  It  was  in  these 
suppurative  changes  that  Rosenbach  first  obtained  a  pure  culture  of  the 
Streptococcus  pyogenes.  It  is  also  found  in  pyaemia.  In  puerperal 
pyaemia  the  streptococci  are  found  in  emboli  in  the  bloodvessels  of 
the  kidneys.  They  are  also  found  in  severe  forms  of  arthritis,  acute 
endocarditis,  in  many  cases  of  secondary  infection  following  scarlet 
fever,  and  in  diphtheritic  false  membranes. 


90  SPECIAL  BACTERIOLOGY 


THE  STREPTOCOCCI  OF  ERYSIPELAS. 

The  presence  of  streptococci  in  erysipelatous  inflammations  of  the 
skin  was  first  observed  by  Koch.  Fehleisen  cultivated  the  cocci 
artificially,  and  proved  their  pathogenic  properties.  According  to 
the  above  authorities  they  are  more  numerous  upon  the  margins  of 
the  erysipelatous  area,  and  may  even  be  seen  in  the  lymph  channels 
a  little  beyond  the  red  margin  which  marks  the  line  of  progress  of 
the  infection. 

Microscopical  Appearances.  —  Micrococci  arranged  in  chains, 
consisting  of  either  a  few  or  many  individual  cocci. 

Motility. — Non-motile. 

Staining  Reactions. — Any  of  the  watery  solutions  of  the  aniline 
dyes  can  be  used.  It  is  also  stained  by  the  Gram  and  Cladius 
methods. 

Biological  Characters— 

On  Gelatine  Plates  small  dots  like  greyish-white  colonies  form,  which 
appear  rnacroscopically  opaque  and  coarse-grained,  and  never  attain  a 
very  great  circumference. 

In  Gelatine  Stab  Cultures  small  white  round  colonies  form  along  the 
inoculation  track. 

On  Agar  Plates  kept  at  incubator  temperature  point-like  colonies 
develop  which  do  not  attain  any  great  size. 

In  Stroke  Cultures  on  Nutrient  Gelatine  or  Agar  small  round  transparent 
fine  dew-drops  develop  along  the  inoculated  part,  which  remain 
isolated. 

In  Bouillon  the  growth  is  better  than  in  the  solid  media  ;  it  forms  a 
ropy  sediment,  which,  when  the  tube  is  shaken,  rises  in  the  fluid. 
Microscopically  examined,  the  sediment  is  found  to  consist  of  long  chains. 
(See  Photomicrograph,  Fig.  27.) 

On  Potatoes  they  may  grow,  according  to  some  authorities,  but  others 
agree  that  no  growth  occurs. 

Patliogenesis. — Rabbits  inoculated  in  the  ear  exhibit  an  erysipe- 
latous inflammation  extending  from  the  point  of  inoculation  to  the 
head  and  neck.  The  temperature  rises,  and  reaches  its  height  in  from 
eight  to  ten  days,  ending  in  recovery. 

Fehleisen  inoculated  cultures  obtained  from  the  skin  of  patients 
with  erysipelas  into  patients  suffering  from  lupus  and  carcinoma,  and 
has  obtained  positive  results,  a  typical  erysipelatous  inflammation 
having  developed  around  the  point  of  inoculation,  after  a  period 
of  incubation  of  from  fifteen  to  sixty  hours.  This  was  accom- 


, 


-27.—  Streptococcus  of  Erysipelas.     Cover-glass  specimen  from 
bouillon  culture.     Cladius  stain.     X  1000. 


;** 


*// 


•^Ri 


Fir;.  L»S.  —  Diplococcus  Pneumonia-  (Fraenkel).  Cover-glass  speci- 
men from  pneumonic  sputum,  showing  capsules.  Johne's 
method.  X  1000. 


\T.  Jinti-hiii,  i-'.n. r.r.s.,  /•//«./«... 


STREPTOCOCCUS  PERNICIOSUS  PSITTACORUM  91 

panied  by  chilly  sensations  and  an  elevation  of  temperature.  Persons 
who  had  recently  recovered  from  an  attack  of  erysipelas  proved  to  be 
immune. 

STREPTOCOCCUS  PERNICIOSUS  PSITTACORUM. 

Found  by  Eberth  and  Wolff  in  parrots  imported  into  Europe,  the 
mortality  being  very  great.  Nodules  were  present  on  the  surface  of 
the  lungs,  spleen,  and  kidneys.  In  the  bloodvessels  of  the  nodules, 
and  in  the  heart's  blood,  medium -sized  cocci  were  found  with  a  ten- 
dency to  form  chains  (it  must,  however,  be  noted  that  parrots  often 
die  of  bird  typhoid  or  chicken  cholera)  which,  with  an  inferior  lens, 
might  be  easily  mistaken  for  cocci.  Further,  parrots  are  also  often 
affected  with  tuberculosis,  and  during  the  course  of  that  disease  mixed 
infection  with  streptococci  might  occur  (Hirsch  and  Kolle). 

The  disease  produced  by  this  organism  is  of  considerable  import- 
ance, as  being  probably  the  source  of  some  obscure  forms  of  lung 
infection  in  man,  which  have  been  traced  to  diseased  parrots  kept  as 
pets. 

DIPLOCOCCUS  OF  PNEUMONIA  (FRAENKEL). 

STREPTOCOCCUS  LANCEOLATUS  (PASTEUR). 

This  organism  occurs  frequently  in  the  exudate  in  pneumonia  and 
secondary  affections  associated  with  that  disease  (pleuritis,  pericarditis, 
peritonitis,  meningitis,  endocarditis,  etc.).  It  is  usually  present  in 
the  sputum  of  pneumonic  patients,  and  it  is  also  found  in  normal 
sputum  of  healthy  individuals. 

Microscopical  Appearances.  —  Spherical  or  oval  cocci,  usually 
occurring  in  pairs,  but  sometimes  forming  chains  of  three  or  four  elements. 
In  stained  specimens  from  the  fibrinous  exudates  of  croupous  pneumonia, 
and  from  the  blood  of  inoculated  animals,  a  capsule  is  visible  surrounding 
the  cocci.  It  is  also  occasionally  seen  on  stained  preparations  from  the 
surface  of  cultures  or  blood  serum.  (See  Photomicrograph,  Plate  IV., 
Fig.  21.) 

Motility. — Non-motile. 

Staining  Reactions. — The  diplococci  stain  readily  with  the  usual 
aniline  stains,  and  by  the  Gram  method,  which  distinguishes  it  from 
Friedlaiider's  bacillus  of  pneumonia,  the  latter  being  decolorized. 

To  demonstrate  the  capsules  in  cover-glass  specimens,  place  the 
specimen  in  1  per  cent,  acetic  acid  for  one  minute,  dry,  and  stain  with 
Ehrlich's  anilin  water  gentian  violet,  or  stain  by  Johne's  method.  See 
Technique,  §  22.  (See  Photomicrograph,  Fig.  18,  stained  by  Johne's 
method.) 


92  SPECIAL  BACTERIOLOGY 

Biological  Characters. — Grows  in  the  presence  of  oxygen,  aerobic, 
but  is  also  a  facultative  anaerobe.  Under  the  latter  conditions  it  retains 
its  vitality  and  virulence  much  longer.  The  minimum  temperature  for 
its  growth  is  22°  C.,  maximum  39 '5°  C.  for  cultures  on  solid  media,  and 
42-5°  C.  for  those  on  liquid  media,  while  the  optimum  temperature  is  35 
to  37°  C. 

On  Gelatine  at  25°  it  develops  fine  delicate  colonies.  The  gelatine 
is  not  liquefied. 

On  Oblique  surface  agar  (which  must  be  only  slightly  alkaline).  On 
Agar  Plates  and  blood  serum  the  diplococci  grow  in  small,  fairly 
granular  dewdrop-like  colonies. 

In  Bouillon  the  growth  exhibits  nothing  characteristic. 

Milk  is  a  favourable  medium,  and  in  some  cultures  coagulation 
results. 

The  diplococci  grow  best  on  media  containing  blood. 

Vitality.  —  Pneumonic  sputum  attached  to  cloths,  air-dried,  and 
exposed  to  diffuse  daylight,  retained  its  virulence  for  rabbits  in  one 
series  of  experiments  for  a  period  of  19  days,  and  in  another  series  for 
55  days.  Exposed  to  direct  sunlight,  the  same  material  retained  its 
virulence  after  twelve  hours'  exposure.  In  agar  cultures  the  diplococci  do 
not  live  long  (four  or  five  days),  but  in  bouillon  their  vitality  is  more 
prolonged.  The  cause  of  the  cultures  dying  is  the  formation  of  lactic 
and  formic  acids.  Neutralizing  the  cultures  with  calcium  carbonate 
causes  them  to  retain  their  vitality  for  months.  Exposure  for  ten 
minutes  at  52°  C.  is  sufficient  for  their  destruction,  and  they  exhibit  very 
slight  resistance  to  the  ordinary  germicides. 

Pathogenesis. — The  diplococci  of  pneumonia  are  pathogenic  for 
rabbits,  guinea-pigs,  and  mice.  Rabbits  infected  subcutaneously  with 
a  fresh  virulent  bouillon  culture  die  in  one  to  two  days  of  a  typical 
septicaemia;  rats  are  less  susceptible  to  infection,  and  chickens  and 
pigeons  are  immune.  Kruse  and  Pansini  also  found  a  sheep  and  a 
horse  immune. 

BACILLUS  OF  PNEUMONIA  (FRIEDLANDER). 

This  organism  is  seldom  found  in  pneumonic  patients.  It  occurs 
either  alone  or  associated  with  other  organisms,  and  is  frequently 
found  in  the  nasal  discharge  in  catarrh,  and  in  otitis  media  acuta. 

Microscopical  Appearances. — The  bacilli  are  much  larger  than 
the  diplococcus  of  pneumonia,  the  minimum  size  being  1  p.  They  are 
arranged  in  diplo-formation  or  in  chains.  A  capsule  is  present  in 
specimens  from  sputum  and  inoculated  animals,  and  it  can  also  some- 
times be  observed  in  specimens  prepared  from  cultures.  (See  Photo- 
micrograph, Fig.  29.) 


«• 

* 

.** 


,<  * 


FIG.  29.— B.  Pneuinonise  (Friedlander).    Cover-glass  specimen,  showing 
capsules,  from  inoculated  mouse.    Johne's  method.     X  1000. 


FIG.  30.     M.  Tetragenus  in  section  of  spleen  of  inoculated  mouse. 
Gram-Giinther  method.     X  500. 


[71.  BoiehUl,  F.R.U.V.S.,  Photo. 


MICROCOCCUS  TETRAGENUS  93 

Motility. — Non-motile. 

Staining  Reactions. — It  stains  easily  with  the  ordinary  aniline  dyes, 
but  not  by  the  Gram  method. 

Biological  Characters. — It  is  aerobic  and  facultative  anaerobic, 
growing  both  at  ordinary  room  and  incubator  temperatures. 

On  Gelatine  Plates  it  forms  small  elevated  porcelain-like  clusters ;  the 
gelatine  is  not  liquefied,  but  eventually  acquires  a  brownish  colour. 

hi  Gelatine  Stab  Cultures  a  typical  nail-shaped  growth  occurs. 

On  Agar  Media  it  forms  a  whitish  coating. 

On  Potatoes  it  develops  a  yellowish-white  coating  which  contains  gas 
bubbles  at  incubator  temperature. 

Media  containing  grape  sugar  undergo  fermentation,  Co2,  H.,  ethylic 
alcohol,  and  acetic  acid  being  formed. 

Milk  is  not  coagulated. 

Vitality. — At  40°  C.  development  ceases,  the  thermal  death-point 
being  about  56°  C.  The  bacilli  retain  their  vitality  in  ordinary  culture 
media  for  a  long  time,  living  for  several  months. 

Pathogenesis. — The  bacillus  of  pneumonia  is  pathogenic  for  mice 
and  dogs,  and  slightly  so  for  guinea-pigs.  It  is  distinguished 
from  the  diplococcus  of  pneumonia  by  rabbits  being  immune.  Sus- 
ceptible animals  are  inoculated  direct  into  the  pleural  and  abdominal 
cavities.  They  can  also  be  affected  by  inhalation  of  dried  pulverized 
cultures.  In  some  cases  pneumonic  lesions  are  formed. 

MICROCOCCUS    TETRAGENUS. 

This  micrococcus  was  discovered  by  Koch  in  1884  in  a  phthisical 
lung  cavity.  Gaffky  made  a  further  study,  and  described  its  patho- 
genic peculiarities  for  various  experiment  animals.  Biondi  also 
found  it  in  human  saliva. 

Microscopical  Appearances. — When  obtained  from  the  animal 
body  it  occurs  mostly  in  groups  of  four  surrounded  by  a  capsule. 

Motility. — Non-motile. 

Staining  Reactions. — It  stains  by  the  Gram  method,  the  proto- 
plasm remaining  stained  while  the  capsule  is  decolorized  ;  also  with 
the  ordinary  aniline  dyes. 

Biological  Characters. — It  grows  best  in  the  presence  of  oxygen 
on  the  usual  media  at  from  35°  to  38°  C.,  and  also  at  20°  C. 

On  Gelatine  Plates  white,  shiny,  prominent,  round  colonies  develop. 

In  Gelatine  Stab  Cultures  it  grows  on  the  surface  as  well  as  along  the 
track  of  the  needle  ;  on  the  surface  it  forms  a  white,  shiny  exuberance. 
The  gelatine  is  not  liquefied. 


94  SPECIAL  BACTERIOLOGY 

On  Agar  it  forms  a  white,  moist,  irregularly  outlined  covering. 
On  Potatoes,  a  shiny,  thick,  irregular  patch  is  formed. 

Pathogenesis. — In  white  mice  and  guinea-pigs  an  abscess  some- 
times forms  instead  of  general  septicaemia.  Grey  mice,  dogs,  and 
rabbits  are  not  susceptible.  Macroscopically,  no  alteration  can  be 
observed  in  the  organs  of  dead  animals,  but  microscopical  examina- 
tion will  reveal  the  presence  of  the  organism.  In  sections  the 
organisms  will  always  be  found  within  the  capillaries.  (See  Photo- 
micrograph, Fig.  30.) 

MICROCOCCUS  GONORRHCEA. 

(Goxococcus — NEISSER). 

This  organism  was  discovered  by  Neisser  in  1879  in  gonorrhoeal 
pus,  and  described  by  him  as  a  '  gonococcus.'  It  was  cultivated  by 
Bumm  in  1885,  and  its  infective  influence  proved  by  inoculation  into 
men.  It  is  constantly  present  in  virulent  gonorrhoeal  discharges, 
generally  in  the  interior  of  the  pus  cells,  or  attached  to  the  surface  of 
the  epithelial  cells. 

Microscopical  Appearances.  —  Cocci  usually  jointed  in  pairs. 
They  are  shaped  like  a  pair  of  kidneys  placed  with  the  hilum  in 
apposition,  or  like  a  coffee  bean,  and  are  separated  by  a  distinct  inter- 
space. The  length  of  the  gonococci  is  0-8  /*  to  1'6  p,  diameter  0'6  to 
0-8  IJL. 

Staining  Reactions. — It  stains  quickly  with  methyl  violet,  gentian 
violet  fuchsin,  not  so  quickly  with  methylene  blue,  which  is,  however, 
the  best  stain  for  demonstrating  its  presence  in  pus.  (For  special 
staining  methods  see  Technique,  §§  16,  17.)  The  results  with  the  Gram 
method  are  negative,  which  enables  it  to  be  distinguished  from  other 
common  pus  cocci.  Though,  according  to  Bumm,  other  diplococci 
sometimes  occur  in  gonorrhoeal  pus  that  do  not  stain  by  the  Gram 
method,  the  most  trustworthy  diagnostic  character  is  that  the  gonococci 
are  found  within  the  pus  cells  sometimes  in  one  or  two  pairs  only, 
frequently  in  considerable  numbers,  and  sometimes  almost  filling  the 
cells.  (See  Photomicrograph,  Plate  IV.,  Fig.  24.) 

Biological  Characters. — The  gonococcus  grows  only  at  37°  C. 

Plate  Culture  Method.  —  The  gonorrhceal  discharge  is  placed  in  a 
tube  with  human  blood  serum  at  40°  C.,  and  two  reductions  made  in  the 
usual  manner  (see  Technique,  §  112)  into  two  other  blood  serum  tubes 
at  40°  C.  In  these  three  tubes  an  equal  quantity  of  2  per  cent,  peptone 
agar,  previously  dissolved  and  cooled  down  to  40°  C.,  is  added,  and  three 
plate  cultures  are  prepared  and  placed  immediately  in  the  incubator. 


MICROCOCCUS  GONORRHCEA  95 

Instead  of  plates,  Petri-dishes  can  be  used.  In  twenty-four  hours  isolated 
gonococci  colonies  appear.  1'he  superficial  colonies  exhibit  a  dark 
punctiform  centre,  from  which  a  delicate,,  finely  granular  coating 
extends  round  about  the  colony ;  the  deeper  colonies  are  greyish-white 
in  colour,  and  possess  an  uneven  appearance,  and  in  two  to  three  days 
acquire  the  shape  of  a  blackberry.  In  re-inoculating  from  the  colonies 
they  are  found  to  consist  of  a  shiny,  tenacious,  compact  mass. 

Stroke  Cultures  on  oblique  solidified  Blood  Serum  Agar  are  prepared  as 
follows : — 1  part  of  fluid  human  blood  serum  at  40°  C.  is  m&ed  with  3 
parts  of  melted  agar-agar,  also  at  40°  C.,  and  placed  in  an  oblique 
position  to  solidify.  The  growth  on  this  medium  is  luxuriant ;  at  first 
isolated  grey  colonies  appear,  which  later  become  moist,  shiny,  tenacious 
slimy  tufts,  and  from  the  margins  a  thin  film-like  coating  extends. 

A  good  liquid  medium  is  prepared  by  mixing  1  part  of  human  blood 
serum  with  2  parts  of  peptone  bouillon.  In  this  medium  the  gonococcus 
forms  a  membrane  on  the  surface,  while  the  medium  itself  remains 
almost  entirely  clear. 

In  preparing  the  culture  media  animal  blood  serum  can  be  used 
instead  of  human  serum,  although  they  do  not  grow  so  well,  but  never- 
theless the  gonococci  grow  very  well  on  swine  blood  serum. 

Pathogenesis. — Bumm  made  inoculations  into  the  healthy  urethra, 
and  in  two  cases,  once  with  a  third  generation  culture  and  once  with 
culture  transferred  for  twenty  successive  generations,  and  in  both  cases 
a  typical  gonorrhoea  developed  as  the  result  of  the  inoculation.  The 
mucous  membranes  of  man  liable  to  gonorrhoeal  infection  are  those  of 
the  urethra,  conjunctiva,  the  cervix  uteri,  and  the  vagina  in  children. 
Inoculations  of  gonorrhoeal  pus  into  the  vagina  or  conjunctival  sac  of  the 
lower  animals,  dogs,  rabbits,  horses,  apes,  are  without  result. 

In  Blenorrhcea  neonatorum,  according  to  Bumm,  after  infection  the 
presence  of  gonococci  may  be  demonstrated  in  the  superficial  epithelial 
cells  of  the  mucous  membrane  and  between  them ;  that  they  soon 
penetrate  to  the  deeper  layers  ;  and  that  by  the  end  of  forty-eight  hours 
the  entire  epithelial  layer  is  invaded  by  the  diplococci,  which  penetrate 
by  way  of  the  connecting  material,  '  Kitt  substance,'  between  the  cells. 
They  also  multiply  in  the  superficial  layers  of  the  connective  tissue,  and 
give  rise  to  an  inflammatory  reaction,  which  is  shown  by  an  abundant 
escape  of  leucocytes  from  the  capillary  network. 

Bacteriological  Diagnosis. — The  microscopical  examination  of 
the  urethral  discharge  is  of  the  greatest  importance.  Cover-glass 
specimens  are  prepared  from  the  suspected  discharge,  air-dried,  fixed 
in  the  flame,  and  stained  with  a  watery  solution  of  methylene  blue. 
The  cocci  and  nuclei  of  the  pus  cells  are  stained  blue  by 'this  method, 
the  cocci  more  intensely  than  the  nuclei.  The  characteristic  form  of 


96  SPECIAL  BACTERIOLOGY 

the  cocci,  their  position  in  the  pus  cells,  and  their  negative  reaction 
with  the  Gram  staining  method,  allow  the  diagnosis  of  gonorrhoea 
to  be  accomplished  with  positive  certainty.  The  absence  of  the 
gonococci  in  microscopical  specimens  of  urethral  discharge  must  be 
accepted  with  caution,  as  the  gonococci  are  sometimes  situated  deep 
in  the  mucosa  of  the  urethra,  and  often  absent  in  the  superficial 
discharge.  It  is  therefore  necessary  in  doubtful  cases  to  irritate  the 
urethra  and  stimulate  the  discharge.  If  the  secretion  after  repeated 
examination,  also  .with  previous  irritation  of  urethra,  is  found  free 
from  gonococci  (numerous  other  cocci  are  usually  present),  then  the 
gonorrhoea  can  be  considered  terminated,  and  only  a  urethritis 
catarrhalis  existent. 

BACILLUS  PYOCYANEUS. 

(Bacillus  of  green  pus.     Microbe  du  pus  bleu.      Boot.  Aeruginosum.) 

This  bacillus  is  found  in  green  or  blue-coloured  pus,  especially  in 
green-coloured  bandages,  from  which  pure  cultures  are  easily  obtained 
with  plate  cultures. 

Microscopical  Appearances. — Small  thin  bacilli  about  the  length 
of  the  bacillus  of  mouse  septicaemia,  but  a  little  thicker,  and  in  cultures 
often  form  small  chains,  occasionally  growing  into  filaments. 

Motility.  —  Strongly  motile,  possessing  only  one  flagellum  (mono- 
tricha). 

Spore  Formation  does  not  exist. 

Staining  Reactions. — It  stains  readily  with  the  ordinary  aniline 
dyes,  but  not  by  the  Gram  method. 

Biological  Characters.  —  This  organism  is  a  facultative  anaerobe, 
growing  both  at  room  and  incubator  temperature. 

On  Gelatine  Plates,  flat,  irregular,  circumscribed  colonies  develop  with 
radiating  borders ;  the  gelatine  is  liquefied  quickly,  the  surrounding 
media  exhibiting  a  green  fluorescence. 

In  Gelatine  Stab  Cultures  the  medium  is  liquefied  quickly. 

On  Oblique  Surface  Agar  a  whitish  coating  is  formed,  and  the  under- 
lying medium  is  coloured  green. 

On  Glycerine  Agar  the  inoculated  medium  exhibits  at  first  a  blue  colour, 
which  gradually  becomes  darker. 

On  Potatoes  a  greenish-yellow  or  brownish  growth  takes  place,  the 
surrounding  surface  being  coloured  green. 

Bouillon  is  clouded. 

Milk  is  coagulated  and  peptonized. 

This  organism  produces  pyocyanin  and  a  fluorescent  green  colouring 


STREPTOCOCCUS  OF  STRANGLES  97 

matter,   formed  only  in  the  presence  of  oxygen,   which  is   soluble  in 
chloroform. 

Pathogenesis. — This  bacillus  is  pathogenic  for  guinea-pigs  and 
rabbits — 1  c.c.  of  a  bouillon  culture  causing  the  death  of  the  animal  in 
from  12  to  36  hours.  Smaller  amounts  do  not  kill  the  animals,  but 
render  them  immune  to  doses  fatal  to  animals  not  previously 
protected.  In  rabbits  inoculated  with  a  culture  of  the  Bacillus 
anthrax  a  fatal  result  may  be  prevented  by  soon  after  inoculating 
the  animal  with  a  pure  culture  of  the  Bacillus  pyocyaneus.  Wood- 
head's  experiments  indicate  that  the  antidotal  effect  is  due  to  the 
chemical  products  of  the  growth  of  the  bacillus  and  not  to  an 
antagonism  of  the  living  bacterial  cells. 

STREPTOCOCCUS    OF    STRANGLES    OF    THE    HORSE 

(SCHUTZ) 

(Ger.  Druse  der  pferde ;  Fr.  Gourme.) 

This  is  an  infectious  catarrh  of  the  upper  passages  of  the  horse, 
with  suppurative  inflammation  of  the  neighbouring  lymph-glands, 
generally  forming  an  abscess.  The  disease  is  often  complicated  with 
metastatic  abscesses  in  other  distant  organs  and  lymph-glands,  the 
virus  being  carried  by  means  of  the  blood  and  lymph  circulation. 
One  attack  gives  immunity  for  years,  perhaps  for  a  lifetime. 

Microscopical  Appearances. — The  Schiitz  streptococcus  is  found  in 
the  pus  obtained  from  the  lymph-gland  abscesses  in  more  or  less  long 
bundles  of  chains.  The  most  significant  formation  is  when  the  cocci  are 
arranged  in  threads  resembling  a  wreath  of  roses,  lying  either  slightly 
bent  or  undulating  between  the  pus  corpuscles  (see  Photomicrograph, 
Plate  IV.,  Fig.  23).  Individual  cocci  in  the  chain  sometimes  appear 
larger  than  the  others. 

Staining  Reactions. — The  streptococci  stain  very  well  with 
fuchsin  and  gentian  violet.  For  pus  preparations  the  Cladius  method 
gives  good  results,  also  the  Gram  method,  the  decolorizing  being 
accomplished  with  a  saturated  alcoholic  solution  of  fluorescin  according 
to  Kiihne's  modification. 

Biological  Characters. — On  Gelatine  the  growth  is  weak,  white 
colonies  forming  on  the  inoculation  stab.  The  gelatine  is  not  liquefied. 

On  Agar-Agar,  inoculated  with  a  drop  of  pus,  numerous  visible  trans- 
parent colonies  about  the  size  of  a  pin-head  develop.  In  stab  cultures 
at  31°  C.,  they  form  a  greyish-white  zone  with  wing-like  projections. 

On  Solid  Blood  Serum  at  37°  the  growth  is  most  luxuriant.  The 

G 


98  SPECIAL  BACTERIOLOGY 

colonies   appear   at   first  as   shiny  grey   drops ;    and   after   this   a    dry 
iridescent  coating  forms. 

In  Bouillon  a  flocculent  white  mass  develops,  finally  forming  a 
sediment  at  the  bottom  of  the  tube. 

Pathogenesis. — Horses  can  be  inoculated  with  pure  cultures, 
abscesses  forming  at  the  point  of  inoculation,  due  to  necrosis  of  the 
tissues.  Inoculation  into  the  mucous  membrane  of  the  nose  causes 
typical  purulent  nasal  catarrh,  with  accompanying  inflammation  and 
enlargement  of  the  lymph-glands.  Mice  are  also  susceptible  to 
infection  when  inoculated,  an  abscess  forming  at  the  point  of  inoculation, 
accompanied  with  metastatic  suppuration  throughout  the  course  of  the 
lymphatic  and  blood  circulations. 

Differential  Diagnosis. — The  disease  is  differentiated  from 
glanders  by  experimentally  inoculating  field-mice,  which  are  highly 
susceptible  to  glanders,  but  immune  to  strangles. 

The  streptococcus  of  strangles  is  not  a  very  resistant  organism,  as 
white  mice  inoculated  with  dried  pus  remain  unaffected. 


STREPTOCOCCUS   OF   CONTAGIOUS   MAMMITIS 
OF   MILCH    COWS. 

This  is  a  special  form  of  mammitis  occurring  in  milch  cows, 
described  by  Nocard  and  Mollerau.  It  passes  rapidly  from  one 
animal  to  the  other.  The  disease  commences  at  the  base  of  the  teats 
in  the  form  of  indurated  lumps,  which  sooner  or  later  invade  the 
whole  organ. 

Microscopical  Appearances. — Round  or  ovoid  micrococci,  1-25  /z 
long  and  1  //,  broad,  occurring  in  long  straight  or  undulating  chains. 
These  characteristic  chains  are  found  in  the  milk  and  in  the  walls  of  the 
excretory  ducts.  They  can  be  stained  by  the  ordinary  dyes,  but  not  by 
the  Gram  method.  The  growth  of  this  organism  in  cultures  is  checked 
by  a  trace  of  boric  acid.  By  means  of  injections  of  100  grains  of  4  per 
cent,  tepid  solutions  of  boric  acid  into  the  teats  of  affected  udders, 
Nocard  and  Mollerau  succeeded  in  arresting  the  extension  of  the  disease. 
The  organism  is  also  destroyed  by  a  3  per  cent,  solution  of  carbolic  acid. 

Biological  Characters. — On  Gelatine  Plates. — Small,  round,  non- 
liquefying  granular  colonies  of  slow  growth. 

In  Gelatine  Stab  Cultures. — In  the  form  of  a  nail. 

On  Agar-Agar  it  grows  badly. 

Bouillon  is  very  soon  clouded. 

Milk  becomes  acid  and  is  coagulated, 


MICROCOCCUS  OF  GANGRENOUS  MAMMITIS  99 

Pathogenesis. — Pure  cultures  introduced  into  the  teats  produced 
the  disease  in  the  cow  and  goat.  The  dogi  cat,  rabbit,  and  guinea- 
pig  remained  unaffected  by  both  intravenous  and  intraperitoneal  in- 
jections. 

The  disease  is  communicated  from  the  diseased  to  healthy  cows  by 
the  hands  of  the  milkers,  which  can  be  prevented  by  disinfection  of 
the  hands  with  3  per  cent,  solution  of  carbolic  acid.  Milk  from  a 
diseased  cow  also  infects  the  milk  from  the  healthy  animals,  rendering 
both  unfit  Jar  human  consumption. 

MICROCOCCUS  OF  GANGRENOUS  MAMMITIS  OF  MILCH 

EWES. 

This  disease,  also  called  Mai  de  pis  araignee,  causes  great  mortality 
in  affected  ewes. 

Microscopical  Appearances. — According  to  Nocard,  a  very  fine 
micrococcus  0-2  /x  in  diameter,  associated  in  groups  of  four  or  more, 
never  in  chains. 

Staining  Reactions. — Stains  by  the  Gram  method. 

Biological  Characters. — It  is  a  facultative  anaerobic  organism. 

On  Gelatine  Plates. — Grows  on  the  surface  in  round,  white,  liquefying 
colonies. 

In  Gelatine  Stab  Cultures  a  funnel-shaped  growth  occurs. 

On  Agar-Agar  an  abundant  yellowish-white  growth  develops. 

Blood  Serum  is  liquefied. 

Milk  becomes  acid  and  is  coagulated  in  twenty-four  hours. 

Cultures  do  not  remain  virulent  unless  renewed  daily. 

Pathogenesis. — Cultures  inoculated  into  the  teats  of  a  ewe 
produce  a  rapidly  fatal  mammitis.  The  goat  is  refractory. 

Inoculations  into  the  ordinary  experiment  animals  cause  only  a 
slight  oedema,  while  in  the  rabbit  an  abscess  forms,  from  which  it 
recovers. 

DIPLOCOCCUS  OF  PLEURO-PNEUMONIA  CONTAGIOSA 
OF  THE  HORSE  (SCHUTZ). 

(Ger.  Breustseuche  der  Pferde.) 

The  Schutz  bacterium  is  a  small,  slightly  ovoid  organism, 
sometimes  possessing  a  capsule,  found  in  the  pulmonary  tissue,  and 
exudative  pleuritis  generally  present  in  contagious  pleuro-pneumonia 
of  the  horse. 

Staining  Reactions.— The  organism  stains  easily  with  gentian 
violet  or  methylene  blue.  The  Gram  stain  only  gives  positive  results 


100  SPECIAL  BACTERIOLOGY 

when  the  discolorization  in  absolute  alcohol  is  not  continued  for  more 
than  fifteen  or  twenty  seconds,  and  in  two  minutes  the  organism  is 
entirely  decolorized.  The  above  reaction  with  the  Gram  method 
differentiates  this  organism  from  Fraenkel's  pneumococcus,  which  stains 
readily  by  the  Gram  method. 

Biological  Characters. — The  diplococcus  grows  on  gelatine  and 
agar  media  at  room  temperature  ;  the  gelatine  is  not  liquefied,  and  the 
growth  in  both  media  exhibits  nothing  characteristic. 

Pathogenesis. — Affect  mice,  guinea-pigs,  pigeons,  and  rabbits, 
but  not  fowls  or  swine.  The  etiological  importance  of  this  organism 
is  established  by  its  constant  and  characteristic  presence  in  the 
pulmonary  tissues  and  effusions  of  affected  horses,  and  by  the  fact 
that  Schiitz  produced  typical  cases  of  the  disease  by  injecting  healthy 
horses  intrapulmonarily  with  the  diplococcus.  The  bacteria, 
according  to  Von  Rust,  are  present  in  the  nasal  discharges  of  affected 
horses.  Fiedler  isolated  the  diplococcus  from  the  blood  of  affected 
animals,  and  produced  the  disease  on  healthy  animals  by  intra- 
pulmonary  injection. 

BOTRIOMYCOSIS. 

Botryomyces  (Bollinger) ;  Discomyces  (Rivolta)  ;  Botryococcus 
ascoformans  (Kitt)  ;  Mykodermoid  (Johne). 

This  parasite  is  found  in  the  indurated  tissue  of  scirrhus  cord  of 
castrated  horses,  and  also  in  some  form  of  fistulous  withers,  tumours 
at  the  point  of  the  shoulder,  and  other  indurations  of  the  skin  and 
subcutaneous  tissue,  also  in  the  connective  tissue  of  the  pelvic  cavity. 
In  the  pus  of  chronic  mammitis  of  the  cow  (Czoker),  and  in  the 
lungs,  ribs,  and  pleural  cavity  ;  also  in  the  spermatic  cord  of  swine. 
MM.  Porcat  and  IT  or  record  four  cases  in  man.  Microscopically  the 
parasite  appears  in  grape  or  mulberry-shaped  masses  of  pale  greyish- 
yellow  bodies,  about  the  size  of  small  grains  of  sand. 

Microscopical  Appearances. — The  individual  colonies  are  formed  of 
granular  clusters  of  symmetrical  grains,  united  by  a  gelatinous  sub- 
stance, and  enveloped  within  a  close-fitting,  transparent,  colourless 
membrane,  which  holds  them  together. 

Staining  Reactions. — The  parasite  stains  by  the  Gram  method, 
and  when  eosin  is  used  for  a  contrast,  it  stains  the  gelatinous  substance. 
Picric  acid  has  a  similar  action. 

Biological  Characters. — When  grown  on  gelatine  and  potato 
media,  the  conglomerated  and  the  envelop  formation  no  longer  exist. 
Gelatine  is  slowly  liquefied  ;  the  developing  colonies  present  first  a  grey 


BACILLUS  ACNES  CONTAGIOS^  101 

colour,  finally  becoming  yellow,  and  consist  of  distinct  individual  cocci. 
(Kitt  considers  the  Botryomyces  to  be  a  variety  of  the  Staphylococcus 
pyogenes  aureus.) 

In  Gelatine  Stab  Cultures  it  forms  greyish-white  threads,  with  slow 
liquefaction  commencing  at  the  periphery. 

On  Potatoes  it  forms  a  flat  yellowish  coating. 

Pathogenesis. — Rabe  and  Kitt  inoculated  horses  with  pure 
cultures,  and  in  four  to  six  weeks  genuine  fibromas  appeared.  Mice 
are  immune.  Sheep  and  goats  exhibit  inflammation  of  the  skin  ;  later, 
necrosis  accompanied  with  oedema.  Guinea-pigs  die  from  septicaemia 
changes. 

The  parasite  generally  confines  its  ravages  to  its  primary  seat. 
It  may,  however,  migrate  toward  the  lymph  glands  and  into  the 
blood,  when  metastatic  foci  appear,  usually  localized  in  the  lungs, 
skin,  etc. 

BACILLUS  ACNES  CONTAGIOS^  OF  THE  HORSE 
(D1ECKERHOFF  AND  GRAWITZ). 

This  disease  is  a  pustular  dermatitis  known  as  contagious  acne  of 
the  horse,  and  is  very  readily  transmitted  to  other  animals.  In 
severe  cases  ulcerations  and  inflammation  of  the  lymph  vessels  and 
glands  occur.  It  is  easily  distinguished  from  farcy  by  the  cicatriza- 
tion of  the  ulcers,  and,  moreover,  the  pustules  do  not  occur  in  farcy. 
It  is  distinguished  from  horse-pox  or  variola  of  the  horse  by  the  fact 
that  in  variola  the  eruptions  are  always  localized  in  the  lips,  nostrils, 
and  pasterns.  It  is  further  identified  by  the  presence  of  the  bacillus 
discovered  by  Dieckerhoff  and  Grawitz  in  the  pustules. 

Microscopical  Appearances. — Very  small  ovoid  bacilli,  2  //,  long, 
occurring  singly  and  also  forming  small  chains. 

Motility . — Non-motile . 

Staining  Reactions. — Somewhat  refractory  with  ordinary  stains, 
but  stains  by  the  Gram  method. 

Spore  Formation . — Ab sent. 

Biological  Characters. — In  Gelatine  8tab  Cultures  white  colonies 
about  the  size  of  a  millet-seed  develop  along  the  course  of  the  needle. 

On  A  gar- A  gar,  white  colonies  develop  very  slowly. 

On  Blood  Serum,  especially  from  horses  and  cattle  (37°  C.),  in  twenty- 
four  hours  small  white  colonies  develop  on  the  surface  of  the  medium,  a 
granular  deposit  being  formed  in  the  water  of  condensation. 

On  Potatoes  it  exhibits  no  growth. 


102  SPECIAL  BACTERIOLOGY 

Pathogenesis. — Affects  the  horse,  rabbit,  guinea-pig,  ox,  sheep, 
dog,  and  mice.  The  disease  can  be  produced  in  horses  by  rubbing 
the  acne  scabs  or  a  pure  culture  of  the  bacillus  into  the  skin. 
Guinea-pigs  subjected  to  similar  treatment  succumb  in  twenty-four 
hours.  Subcutaneous  injection  in  dogs  and  rabbits  produces  toxic 
symptoms  and  death,  but  the  bacilli  do  not  spread  over  the  body. 
Mice  and  field-mice  are  not  affected  by  rubbing,  but  by  subcutaneous 
inoculation,  die  in  one  to  ten  days  with  the  formation  of  abscesses, 
the  bacilli  being  found  in  clusters  in  the  organs.  The  natural  disease 
is  transmitted  by  means  of  grooming  utensils,  harness,  blankets,  etc., 
and  is  often  localized  in  the  regions  covered  by  the  saddle  and  the 
girth. 

Tizzoni  and  Giovannini  mention  a  case  of  acne  contagiosa  in  man 
which  proved  fatal  in  thirteen  days,  and  from  the  blood  and  skin 
they  isolated  a  bacillus  morphologically,  and  in  cultures,  resembling 
the  bacillus  of  mouse  septica?mia,  but  it  was  not  pathogenic  for  mice, 
while  it  produced  fatal  results  with  rabbits  and  guinea-pigs,  the 
conditions  being  similar  to  those  found  in  the  man.  They  considered 
this  organism  as  only  of  secondary  importance,  and  that  the  Staphylo- 
coccus  pyogenes  was  the  cause  of  the  acne. 


THE  STREPTOTHRICES. 

The  organisms  belonging  to  this  subdivision  resemble  in  their 
structure  at  one  time  the  thread  fungi,  and  at  other  times  the 
bacteria.  Like  the  mould  fungi,  they  form  cylindrical  threads  out  of 
round  cells  which  branch  dichotomously,  finally  becoming  visible  to 
the  naked  eye  as  irregular  radiating  thread  masses  or  mycelia.  Single 
threads  or  fruit  hyphae  grow  upwards  out  of  the  substance,  free  in  the 
air,  and  break  up  into  chains  of  round  germ  cells  (spores  or  conidia), 
which  detaching  from  the  plant  are  carried  by  the  air,  and  the 
preservation  of  the  species  is  thus  assured. 


STREPTOTHRIX    ACTINOMYCES    BOVIS. 

The  true  nature  of  Actinomyces  bovis  was  first  recognised  by 
Bellinger  in  cattle  in  1877.  The  disease  is  characterised  by  the 
formation  of  specific  tumours  tending  to  suppuration,  the  lesions 
being  frequently  located  in  the  jaw  bones  and  subcutaneous  con- 
nective tissue  of  the  maxillary  region,  and  are  commonly  known  as 
wen,  osteosarcoma,  lumpy-jaw,  etc.,  and  when  affecting  the  tongue  as 


STREPTOTHRIX  ACTINOMYCES  BOVIS  103 

wooden  tongue,  owing  to  the  indurated  condition  of  that  organ.  It 
also  occurs  in  the  retropharyngeal  lymph-glands  (Clyers). 

The  author  recorded  in  the  Veterinary  Journal  a  case  in  the 
parotid  gland  of  a  young  cow  in  California,  which  finally  obliterated 
all  the  bloodvessels,  the  diseased  gland  being  successfully  extirpated. 
The  disease  has  also  been  found  in  the  liver,  nasal  cavities,  larynx, 
lungs,  and  vertebrae.  In  the  pig  the  muscles,  lungs,  mammae,  and 
bones  of  the  cervical  and  dorsal  vertebrae  are  affected ;  a  case  has  also 
been  recorded  in  the  dog ;  and  cases  of  actinomycosis  of  the  tongue 
in  the  horse  are  also  mentioned. 

Section  of  a  specific  tumour  reveals  an  abundance  of  granulation 
tissue,  studded  with  soft  parts  or  nodules  of  various  sizes,  containing 
numerous  yellow  or  occasionally  colourless  grains,  the  smallest 
appearing  about  the  size  of  a  grain  of  sand ;  the  larger,  due  to  the 
union  of  the  smaller  grains,  are  of  different  forms.  The  grains  vary 
in  size  from  01  to  1  mm.  or  more,  being  frequently  cretaceous. 
The  above  -  mentioned  yellow  granules  are  characteristic  of  an 
actinomycotic  tumour.  Ponfick  transmitted  the  disease  to  other 
animals  by  means  of  those  granules. 

Microscopical  Appearances. — Examined  under  a  low  power,  the 
unstained  granules  appear  as  dark,  finely  granular,  round  or  irregular 
balls.  Under  a  high  power  and  after  the  specimen  is  stained,  the 
organism  is  found  to  consist  of  a  central  zone  of  very  fine  filaments, 
ramified  and  intermingled  in  a  close  network,  with  a  few  cocci  arranged 
in  the  centre,  and  a  peripheral  zone  consisting  of  radiating  pyriform 
elements  with  large  swollen  or  club-shaped  extremities,  which  are  either 
simple  or  branching.  The  branches  are  given  off  from  either  the 
pedicle  or  the  club,  and  sometimes  by  subdividing  themselves,  the  whole 
resembling  the  capitulum  of  a  daisy  (see  Photomicrograph,  Fig.  32). 

There  are  other  forms  of  actinomyces  which  are  quite  small,  the 
club-like  enlargements  being  absent,  and  Cornil  and  Babes  describe  a 
special  condition  of  the  filaments  of  the  periphery  terminating  in  various 
slight  enlargements  bearing  conidia.  The  threads  sometimes  undergo 
segmentation,  and  resemble  threads  of  bacilli.  Finally,  an  agglomera- 
tion of  club  forms,  consisting  of  masses  of  cocci,  are  sometimes  observed. 

Unstained  specimens  are  prepared  by  squeezing  the  material  under 
the  cover-glass. 

Staining  Reactions.— It  stains  best  by  the  Gram  method,  especially 
by  Giinther's  modification  (see  Technique,  §  38),  also  with  heated  carbol 
fuchsin.  Double  staining  can  be  obtained  with  the  Gram  method  and 
picro-carmine  or  saffronine,  the  threads  of  the  fungi  being  stained  a 
blue-black  colour  by  the  Gram  method,  while  the  clubs  are  stained  red 
with  carmine. 


104  SPECIAL  BACTERIOLOGY 

Biological  Characters. — The  actinomyces  is  a  typical  streptothrix 
possessing  all  the  characteristics  peculiar  to  that  species.  It  is  anaerobic 
and  facultatively  aerobic.  Some  authorities  state  that  the  growth  is 
best  under  anaerobic  conditions  when  first  obtained  from  the  animal 
body,  while  others  consider  it  grows  best  aerobic. 

On  Gelatine  Plates,  in  six  days  a  very  limited  greyish-yellow  growth 
develops,  sometimes  on  the  surface  and  sometimes  in  the  depth  of  the 
medium. 

In  Gelatine  Stab  Cultures. — At  the  commencement  a  du)l  yellowish- 
white,  elevated,  shining  compact  growth  develops  on  the  surface  of  the 
medium,  sinking  later  from  slight  liquefaction  of  the  gelatine  ;  along  the 
track  of  the  needle  small  yellowish-white  knots  develop  at  first,  bristly 
outgrowths  appearing  later. 

On  Plain  and  Glycerine  Agar  opaque  nodules  about  the  size  of  a  pin- 
head,  which  remain  isolated  for  weeks  or  months,  develop,  the  peripheries 
being  formed  of  a  fine  delicate  network.  Large  white  nodules  about 
the  size  of  a  lentil  (rosette  forms)  sometimes  also  develop  (see  Photo- 
graph, Fig.  31). 

On  the  oblique  surface  of  Blood  Serum  the  individual  granules  exhibit  a 
yellowish  brick  or  red-rose  colour,  covered  with  whitish  downy  filaments 
or  threads. 

In  Bouillon  the  medium  is  not  clouded,  but  round  masses  are  formed 
in  the  bottom  of  the  tube,  which  are  separated  with  difficulty  by 
shaking. 

In  Milk  a  granular  growth  takes  place,  followed  by  gradual  peptonising. 

On  Potatoes  the  growth  presents  a  yellowish-red  colour,  covered  with 
a  downy  mass  of  threads. 

In  Eggs. — It  grows  well  in  pigeon  and  hen's  eggs,  either  raw  or  when 
cooked  for  three  or  four  minutes  (prepare  the  egg  and  inoculate  accord- 
ing to  Giinther's  method — see  Technique,  page  52).  The  prepared  egg  is 
placed  in  the  incubator  with  the  inoculated  end  uppermost,  and  exa- 
mined in  from  nine  to  twenty-eight  days ;  if  no  putrefaction  has  taken 
place  or  decolorization,  the  growth  appears  in  raw  eggs  both  in  the 
white  and  in  the  yelk,  and  cloudy  masses  of  slime  resembling  nasal 
mucus  develop  in  the  albumen.  In  cooked  eggs  opaque  white  spots  of ' 
the  size  of  pin-heads  develop  between  the  yelk  and  white,  and  finally  a 
mass  is  formed  in  the  track  of  inoculation  and  on  the  surface  of  the 
coagulated  albumen. 

Microscopical  Examination  of  Cultures. — The  growths  on  agar 
media  consist  mostly  of  short  rods  mostly  straight,  but  often  comma 
shaped,  or  sometimes  further  bent.  The  dimensions  of  the  rods  vary ; 
they  may  appear  plump  and  thick,  very  slender  and  short,  or  in  long 
thick  rods,  and  sometimes  club  or  olive-shaped  at  the  ends  (see  Photo- 


Fi<;.  31.  —  Actinomyces 
Bovis.  Glycerine-agar  cul- 
ture. '  Rosette '  form. 


Fie.  .T_'.— Actinomyces  Bovis.    Section  of  a  tumour  from  the  jaw. 
Stained  by  the  Gram-GUnther  method.     X  45P. 


Fie.  33. — Actiuomyces  Bovis.     Cover-glass  specimen  from  agar 
culture, 'showing  clubs.'    Fuchsin.     X  1000. 

[T.  Howhiii,  !•'.];.<'.  r. s..  rii»t< 


i,  189S. 


STREPTOTHRIX  ACTINOMYCES  BOVIS  105 

micrograph,  Fig.  33).  Undulating  or  spiral  rods  are  sometimes  observed, 
but  rarely  in  agar  cultures. 

In  Egg  Cultures  beautiful  net-like  masses  of  threads  occur ;  the  threads 
at  the  periphery  of  the  mass  radiating,  the  ends  being  sometimes  swollen 
and  club-shaped. 

The  threads  and  also  the  short  rods  stain  both  by  the  Gram  method 
and  with  fuchsin.  When  the  threads  are  stained  one  hour  in  water 
fuchsin  segments  are  sometimes  observed,  consisting  of  long  and  short 
rods,  and  short  coccoid-like  bodies  arranged  in  an  irregular  manner,  and 
separated  by  unstained  interspaces  of  various  breadths.  Finally,  micro- 
coccus-like  bodies  are  also  found  in  agar  and  egg  cultures,  isolated  in 
clusters  of  various  sizes.  They  are  sometimes  clubbed  or  oval,  some- 
times more  irregular  and  angular,  staining  intensely  with  gentian 
violet  and  by  the  Gram  method.  Inoculated  in  fresh  media  they  give 
rise  to  fresh  rods  and  threads. 

Vitality. — It  remains  living  in  cultures  from  nine  to  twelve  months. 
It  is  killed  by  heating  five  minutes  at  75°  C. 

Pathogenesis. — Johne  transmitted  the  disease  to  the  cow  and 
the  calf  by  subcutaneous  intraperitoneal  and  mammary  inoculation. 
Ponfick  and  Israel  also  transmitted  it  to  the  calf  and  to  the  rabbit. 
Israel  made  his  inoculation  with  the  actinomycosis  of  man,  the 
results  being  the  same  as  those  obtained  with  tumours  from  cattle. 
Accidental  contamination  is  also  recorded  in  persons  attending  affected 
animals. 

The  disease  is  supposed  to  be  communicated  to  susceptible  animals 
(Omnivora  and  Herbivora)  by  means  of  their  food,  especially  straw 
and  barley  husks.  Johne  found  an  identical  fungus  on  the  surface 
of  husks  of  barley,  arrested  on  the  tonsils  of  a  healthy  pig.  Piana 
also  discovered  vegetable  debris  accompanying  the  actinomyces  in  a 
tumour  in  the  tongue.  In  a  case  of  abdominal  actinomyces  in  the 
human  subject,  Ammentorp,  in  opening  one  of  the  abscesses,  found  in 
its  centre  a  pin- sized  concretion,  in  the  middle  of  which  a  piece  of 
barley  awn  was  visible  about  2  cm.  long.  A  similar  case  was  also 
observed  in  the  Clinic  at  Vienna.  In  this  instance  the  patient  also 
suffered  from  abdominal  actinomycosis,  and  a  faecal  stone  which  was 
found  in  the  patient  contained  at  its  centre  a  barley  awn.  Nocard 
records  a  case  in  a  stableman  kicked  by  a  horse  where  a  phlegmon 
developed  in  the  upper  part  of  the  thigh,  in  which  microscopic 
examination  revealed  the  characteristic  tufts  of  the  actinomyces. 
The  actinomyces  affecting  man  is  distinguished  from  that  of  cattle 
by  its  tendency  to  form  tumours,  and  the  slow  manner  in  which  the 
disease  spreads  in  the  surrounding  structures,  the  newly  formed 


106  SPECIAL  BACTERIOLOGY 

granulation  tissue  degenerating  more  quickly ;  suppuration  also  being 
more  pronounced,  accompanied  by  the  formation  of  fistulae  which 
undermine  the  skin,  passing  through  the  muscular  tissue,  and  by  this 
means  the  fungus  spreads  from  the  jaws  or  neck  to  the  pleura  and 
ungs,  and  through  the  diaphragm  into  the  peritoneal  cavity. 

ACTINOMYCES   MUSCULORUM   SUIS. 

This  organism  was  discovered  by  Diincker  in  1884,  in  the  muscles 
of  swine.  Although  resembling  the  Actinomyces  bovis,  it  is  not 
identical  (Giinther). 

STREPTOTHRIX   HOFFMANI. 

This  is  another  organism  very  similar  in  its  growth  to  the 
Actinomyces  bovis.  It  is  found  in  the  air. 

Microscopical  Appearances. — It  occurs  as  a  branched  mycelium, 
with  swollen,  club-like  ends. 

Staining  Reactions. — It  is  stained  by  the  Gram  method. 

Biological  Characters. — It  is  aerobic,  growing  only  at  a  tempera- 
ture of  over  22°  C. 

On  Agar  Media  it  grows  in  light  brown  warty  colonies,  which  very 
soon  coalesce. 

Bouillon  remains  clear ;  a  thick  sediment  is  formed. 

Potatoes — There  is  no  growth. 

Pathogenesis. — For  guinea-pigs  and  rabbits,  only  by  subcutaneous 

injections  ;  a  local  abscess  being  formed,  and  in  the  pus  actinomycotic- 

like  tufts  with  club-shaped  ends  are  sometimes  present,  distinguished 

from  the  Actinomyces  bovis  by  the  clubs  staining  the  same  as  the  threads. 

STREPTOTHRIX  ACTINOMYCES  HOMINIS   (ISRAEL). 

This  organism  was  discovered  by  Israel  and  Wolff  in  two  cases  of 
human  actinomycosis,  and  in  its  macroscopical,  microscopical,  and 
staining  reactions  resembles  the  Actinomyces  bovis. 

The  differentiation  in  cultures  is,  however,  well  marked,  as  it 
only  grows  luxuriantly  under  anaerobic  conditions,  and  under  aerobic 
slightly  or  not  at  all.  The  optimum  temperature  is  37°,  and  the 
development  is  very  slow. 

On  Agar-Agar  irregular  opaque  colonies  are  formed,  which  in  seven 
days  attain  the  size  of  a  pin-head ;  usually  coalescing.  To  obtain  pure 


STREPTOTHRIX  MADURA  107 

cultures,  remove  the  pus  under  sterile  precautions,  and  wash  the  small 
nodules  or  grains  it  contains  in  sterile  water,  and  place  them  deep  down 
in  a  tube  of  agar.  As  it  develops,  a  thin  film  forms  round  the 
periphery,  from  which  further  nodules  sometimes  develop.  The  nodules 
sometimes  attain  the  size  of  a  lentil,  and  rosette  forms  appear. 

Bouillon  remains  clear ;  a  sediment  consisting  of  white  scales  forms. 

In  Raw  Eggs  a  cloudy  slime  forms. 

In  Cooked  Eggs  a  greasy,  granular  mass. 

In  the  cultures,  usually  straight,  sometimes  bent,  rods  with  slightly 
swollen  ends  (somewhat  like  the  bacillus  of  diphtheria)  are  found.  The 
filament  masses  are  present,  as  a  rule,  only  in  egg  cultures. 

Pathogenesis. — Affects  guinea-pigs  and  rabbits  by  intraperitoneal 
inoculation  ;  a  genuine  actinomycosis  resulting,  with  the  formation  of 
typical  tumours. 

The  differential  diagnosis  between  this  and  Actinomyces  bo  vis 
seems  to  exist  only  in  culture  peculiarities. 

STREPTOTHRIX    MADURA    (VINCENT) 

Found  in  madura  foot,  an  ulcerative  affection  of  the  feet,  rarely 
of  the  hands,  in  the  East  Indies ;  also  in  America,  Morocco,  and 
Italy. 

Microscopical  Appearances. — The  parasite  consists  of  branched 
threads,  1  to  1'5  p  thick,  which  on  the  surface  of  some  media  present 
filaments  growing  upwards,  but  spores  are  also  formed  in  the  substance 
of  the  medium. 

Staining  Reactions. — The  threads  and  spores  stain  with  the  usual 
aniline  dyes  and  by  the  Gram  method. 

Vitality. — The  threads  are  destroyed  by  heating  for  three  to  five 
minutes  at  60°  C.,  the  spores  at  85°  C. 

Biological  Characters.  —  Strongly  aerobic ;  grows  at  ordinary 
temperature,  but  best  at  37°  C. 

On  Agar  the  growth  is  limited,  while  on  Glycerine  Agar  it  is  luxuriant. 

Gelatine  is  not  liquefied. 

The  developing  colonies  are  nodular,  hard,  yellowish-white  in  colour, 
later  becoming  reddish. 

There  is  no  growth  on  serum  or  in  eggs. 

In  Bouillon  the  growth  is  limited,  granules  developing  slowly. 

On  Acid  Potatoes. — Warty,  at  first  white,  later  red  or  orange  coloured 
exuberances,  studded  with  white  threads. 

Milk  is  peptonised  slowly. 

The  growth  is  also  very  luxuriant  on  slightly  acid  vegetable  infusions. 


108  SPECIAL  BACTERIOLOGY 

Pathogenesis. — Inoculation  of  various  animals  has  failed,  only 
local  reactions  being  manifested. 


STREFrOTHRIX    EPPINGER. 

This  organism  was  found  by  Eppinger  in  a  brain  abscess.  It 
consists  of  a  branched  mycelium.  Fruit  hyphae  and  spores  are  only 
found  in  potato  cultures. 

Staining  Reactions. — It  stains  by  the  Gram  method. 

Biological  Characters. — It  is  aerobic,  growing  best  at  37°  C. 
On  Gelatine,  yellow  warty-shaped    elevated  colonies  develop,  which 
do  not  liquefy  the  gelatine. 

On  Glucose  Agar  the  growth  is  orange  coloured. 
On  Potatoes  it  forms  a  yellowish-red  coating. 
Bouillon  remains  clear,  but  flaky  lumps  develop. 

Pathogenesis. — Guinea-pigs  and  rabbits  inoculated  with  this 
organism  develop  a  pseudo-tuberculosis. 


STREPTOTHRIX  FARCINICA  (ROSSI  DORIA). 

(Fr.  Bacille  du  farcin  du  breufs  (Nocard) ;  Eng.  Bovine  Farcy.) 

This  bovine  malady  was  formerly  very  prevalent  in  France,  and 
exists  also  in  Guadeloupe.  The  lesions  are  usually  located  in  the 
limbs,  and  consist  of  swelling  of  the  lymph  vessels,  terminating  at  the 
corresponding  lymph-glands,  those  most  usually  affected  being  the 
brachial,  pectoral,  and  prepectoral  groups,  which  suppurate  slowly. 
When  the  abscesses  are  opened  the  animal  seems  to  recover,  but  other 
tumours  appear,  the  animal  pining  and  dying  of  general  marasmus. 
The  autopsy  shows  pseudo-tubercular  lesions  with  purulent  centres 
in  the  lungs,  liver,  spleen,  and  lymph-glands. 

Microscopical  Appearances. — Small,  interwoven  masses  of  threads, 
about  0*25  ^  thick,  arranged  in  tufts,  are  present  in  the  pus  from  the 
abscesses. 

Staining  Reactions. — It  stains  by  the  Gram  method,  but  is  de- 
colorized when  the  contact  with  the  alcohol  is  prolonged  ;  it  also  stains 
with  Weigert's  double  stain.  The  spores  stain  with  difficulty. 

Biological  Characters. — It  is  a  purely  aerobic  organism,  growing 
best  between  30°  to  40°  C. 

In  Bouillon  it  forms  round  pellicles  of  a  dull  grey  colour  and  oily 
appearance,  floating  in  the  liquid  with  an  abundant  film  on  the  surface. 


BACILLUS  ANTHRACIS  109 

On  Agar  and  Gelatine,  small,  more  or  less  rounded  opaque  masses, 
thicker  at  the  periphery  than  in  the  centre,  develop. 

In  Milk  it  grows  without  changing  the  reaction  or  causing 
coagulation. 

On  Potatoes  the  growth  is  rapid. 

Pathogenesis. — Pure  cultures  or  pus  injected  into  the  peritoneal 
cavity  of  a  guinea-pig  cause  pseudo-tuberculosis  of  the  peritoneum  in 
nine  to  twenty  days,  the  characteristic  tufted  organisms  being  present 
in  the  centre  of  the  nodules.  Intravenous  inoculation  causes  a 
generalized  pseudo-tuberculosis.  Intravenous  inoculation  of  cattle 
and  sheep  causes  a  slow-forming  pseudo-tuberculosis. 

Subcutaneous  inoculation  in  refractory  animals  causes  an  abscess 
which  heals  quickly. 


BACILLUS   ANTHRACIS. 

(Ger.  Milzbrand  bacillus ;  Fr.  Bacteridie  du  charbon). 

This  organism  is  always  present  in  the  blood  of  animals  affected 
with  anthrax,  and  can  be  isolated  in  pure  cultures  on  artificial 
media.  When  susceptible  animals  are  inoculated  with  portions  of 
pure  cultures,  conditions  similar  to  those  found  in  the  animal  from 
which  the  original  cultures  was  obtained  are  produced. 

Microscopical  Appearances. — In  the  blood  of  animals  recently 
dead,  the  bacilli  occur  as  large  rods  of  variable  size,  from  3  to  10  /z  long 
and  1  to  Ij  fji  broad,  often  arranged  in  threads  formed  by  several  rods 
jointed  together  (see  Photomicrograph,  Fig.  34).  In  unstained  specimens 
examined  by  means  of  a  hanging  drop,  the  ends  of  the  rods  appear  round  ; 
while  in  stained  specimens  the  ends  of  the  rods  are  square.  Under  a  high 
magnification  the  ends  are  found  to  be  a  trifle  thicker  than  the  body  of 
the  bacillus,  and  occasionally  somewhat  indented  and  concave — compared 
by  Fraenkel,  when  stained  with  methylene  blue,  to  small  pieces  of 
bamboo  cane. 

Bacilli  obtained  from  the  blood  of  affected  animals  possess  capsules 
(see  Photomicrograph,  Plate  I.,  Fig.  1,  also  Fig.  35).  To  demonstrate 
the  capsules,  stain  the  specimens  by  Johne's  special  process  (see 
Technique,  §  22).  Capsules  can  also  be  demonstrated  when  the 
bacilli  are  cultivated  in  liquid  blood  serum  medium.  Giinther  has  also 
found  the  capsules  present  in  bacilli  in  sections  stained  with  methylene 
blue.  The  square  ends  and  the  presence  of  capsules  help  to  distinguish 
the  Bacillus  anthracis  from  certain  other  organisms  that  resemble  it 
morphologically,  especially  putrefactive  bacteria. 

After  death,  the  bacilli  are  only  to  be  found  in  bloodvessels.     In 


110  SPECIAL  BACTERIOLOGY 

sections  of  organs  stained  by  the  Gram  or  Cladius  methods  they  are 
easily  demonstrated  (see  Photomicrograph,  Plate  I.,  Fig.  .3),  showing  a 
section  of  a  mouse's  lung  with  the  capillaries  filled  with  bacilli. 

On  artificial  media  the  bacilli  grow  in  long,  parallel,  or  somewhat 
twisted  and  interlaced  threads,  which  either  form  spores  or  degenerate 
into  the  so-called  involution  forms  (see  Photomicrograph,  Fig.  37). 

Motility. — Non-motile. 

Staining  Reactions. — The  bacilli  stain  easily  with  all  the  ordinary 
aniline  dyes,  with  haematoxylin,  and  by  the  Gram  and  Cladius  methods. 

Spore  Formation. — The  bacilli  form  spores  under  aerobic  conditions 
at  a  temperature  of  15°  to  as  high  as  37°  C.  Giinther  considers  28°  C. 
the  optimum,  and  that  at  a  higher  temperature  the  formation  is  some- 
what irregular. 

Spores  are  never  formed  in  the  living  animal  or  in  unopened  carcases.  The 
latter  is  most  important  from  a  sanitary  point  of  view,  in  regard  to  the 
disposal  of  the  carcases  of  animals  dead  from  anthrax.  The  spores  are 
ovoid,  and  one  to  two  times  as  long  as  broad,  one  spore  being  present  in 
nearly  every  bacillus,  giving  the  thread  the  appearance  of  a  chain  of 
beads  (see  Photomicrograph,  Plate  I.,  Fig.  2).  For  the  special  method 
of  staining  the  spores  of  the  Bacillus  anthracis,  see  Technique,  §  27. 

Vitality. — The  resistance  of  anthrax  spores  to  outside  influences  is 
not  always  constant.  Some  spores  are  killed  by  exposure  to  5  per  cent, 
carbolic  acid  in  two  days,  and  to  steam  at  100°  C.  in  three  minutes, 
while  others  resist  5  per  cent,  carbolic  acid  over  forty  days,  and  steam  at 
100°  C.  for  more  than  twelve  minutes.  In  a  dry  state  the  spores  are 
destroyed  instantly  at  160°  C. 

When  the  bacilli  are  cultivated  in  bouillon  to  which  ^JL^  to  TJQn 
bichromate  of  potash  is  added,  they  lose  the  faculty  of  forming  spores 
without  losing  their  virulence.  The  power  of  forming  spores  is  also  lost 
when  the  bacilli  are  cultivated  for  many  generations  on  gelatine  media. 

Biological  Characters. — The  Bacillus  anthracis  is  a  facultative 
aerobic  organism  growing  best  in  the  presence  of  oxygen,  and  in  its 
absence  slowly  without  liquefying  the  media.  Exhibits  no  growth  in 
CO,,.  It  grows  quickly  at  37°  C.,  and  ceases  to  grow  under  12°  and  above 
45°  C. 

On  Gelatine  Plates.— On  the  surface  small  whitish  colonies  appear, 
while  those  deeper  in  the  medium  are  of  a  greenish-black  colour.  Under 
a  low  power  the  colonies  exhibit  a  characteristic  tangled  mass  of  single 
threads  projecting  beyond  the  edges  of  the  colonies  in  curly  hairy  tufts. 
The  colonies  begin  to  liquefy  in  three  or  four  days. 

In  Gelatine  Stab  Cultures. — In  twelve  to  twenty  hours  a  thick,  white 
central  thread  appears,  from  which  white  threads  and  branching  rootlets 
radiate  (see  Photo.,  Fig.  36).  After  two  days,  liquefaction  commences  on 


FIG.  34.— B.  Anthracis.     Leptothrix  formation  in  a  virulent  bouillon 
culture.     Methylene  blue.     X  1000. 


Fio.  35. — B.  Anthracis,  with  capsules  in  mouse's  blood. 
Stained  by  Johne's  method.     X  1500. 


FIG.  36.— B.  Anthracis.     Stab  culture 
in  gelatine. 


[T.  Boii'hitt,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


BACILLUS  ANTHRACIS  111 

the  surface  of  the  medium,  spreading  outwards  and  downwards  until  the 
whole  medium  is  eventually  liquefied,  and  the  bacterial  mass  sinks  to  the 
bottom  of  the  tube. 

On  Agar  Plates  the  growth  is  similar  to  that  on  gelatine  plates, 
but  the  colonies  are  not  so  compact,  and  consist  of  masses  of  long 
threads  matted  together,  the  growth  continuing  upwards  upon  the 
surface  of  the  medium. 

On  oblique  surface  Agar  a  greyish-white  tenacious  coating  is 
formed  with  thready  edges,  the  condensation  water  remains  clear,  or  is 
only  slightly  clouded. 

Blood  Serum  is  liquefied  slowly. 

Bouillon  remains  clear,  but  a  cloudy  sediment  is  formed. 

Milk  is  coagulated  and  afterwards  peptonized. 

On  Potato  it  develops  as  a  dry  granular  whitish  covering,  more  or  less 
limited  to  the  point  of  inoculation. 

Pathogenesis. — Affects  man,  cattle,  sheep,  horses,  guinea-pigs, 
rabbits,  mice,  and  swine,  and  dogs  under  exceptional  circumstances,  the 
primary  localization  usually  being  in  the  throat ;  rats  are  also  difficult  to 
infect.  In  man  the  disease  is  named  according  to  the  manner  of  in- 
fection. 

1.  Pustula  Maligna.  —  This  is  the  most  common   form,   and  is  the 
result  of  accidental  inoculation  of  a  cutaneous  wound. 

2.  Pulmonary  form. — Woolsorters'    and    ragpickers'    disease   is   the 
result  of  the  inhalation  of  dust  charged  with  anthrax  spores. 

3.  Bowel  or  Intestinal  Anthrax. — Due  to  the  consumption  of  meat  from 
anthrax  carcases. 

Inoculation  into  Animals. — Portions  of  a  pure  culture  of  the 
Bacillus  anthracis,  when  introduced  into  the  subcutaneous  tissues  of  the 
abdominal  wall  of  guinea-pigs  or  rabbits,  cause  the  death  of  these 
animals  in  forty-eight  hours.  Little  or  no  change  can  be  observed  at 
the  point  of  inoculation,  but  the  subcutaneous  tissue  for  some  distance 
over  the  abdomen  and  thorax  will  be  found  oedematous,  with  small 
ecchymoses  scattered  throughout  the  osdematous  portion  ;  the  under- 
lying muscles  are  pale  in  colour.  The  internal  viscera  show  no  marked 
macroscopical  changes,  except  the  spleen,  which  is  enlarged,  dark- 
coloured,  and  soft.  The  liver  may  present  the  appearance  of  cloudy 
swelling.  The  lungs  are  red  or  pale  red  in  colour,  while  the  heart 
is  usually  filled  with  blood.  The  disease  is  a  true  septicaemia,  and  after 
death  the  capillaries  throughout  the  body  always  contain  the  typical 
rod-shaped  organisms  in  larger  or  smaller  numbers. 

Protective  Inoculation  against  anthrax  is  practised  in  animals, 
according  to  Pasteur's  method,  with  two  vaccines  prepared  from  virulent 
cultures  attenuated  by  cultivation  between  42°  and  43°  C.— - 


112  SPECIAL  BACTERIOLOGY 

No.  I.  Vaccine  grown  fifteen  to  twenty  days  at  42°  to  43°  C. 
No.  II.  „  ten   to   twelve  „  „ 

In  the  preliminary  tests  vaccine  No.  I.  killed  only  mice,  while  vaccine 
No.  II.  killed  both  mice  and  guinea-pigs,  but  not  rabbits. 

Cattle  and  sheep  receive  an  injection  of  0'33  c.c.  of  a  four  days' 
old  bouillon  culture  of  No.  I.,  and  the  same  dose  of  vaccine  No.  II.  in  ten 
or  twelve  days.  The  effects  of  the  inoculation  with  vaccine  No.  I. 
should  be  scarcely  noticeable,  causing  neither  constitutional  nor  local 
symptoms,  while  vaccine  No.  II.  may  or  may  not  cause  some  constitu- 
tional disturbance,  and  when  it  does  the  symptoms  are  rarely 
of  an  exaggerated  nature,  and  disappear  in  a  short  time  if  the  vaccines 
have  been  properly  prepared  and  tested  before  use. 

The  above  vaccines  render  sheep  and  cattle  immune  to  inoculated 
anthrax,  but,  according  to  Koch,  against  natural  infection  by  means  of 
the  intestinal  tract,  i.e.,  bowel  anthrax.  Pasteur's  vaccines  cannot  be 
employed  with  certainty,  and  furthermore,  a  great  many  unsatisfactory 
results  have  occurred,  showing  that  the  strength  of  the  vaccines  cannot 
be  regulated  with  absolute  certainty. 

The  part  played  by  insects  in  conveying  anthrax  has  recently  been 
investigated  by  Dr  Nuttall.  He  shows  that  bed-bugs  and  fleas  may  be 
gorged  on  anthrax  victims,  and  then  placed  on  sound  animals,  which,  on 
being  bitten,  show  no  disease,  while  at  the  same  time  cultivation  and 
inoculation  experiments  made  from  the  above  insects,  as  well  as 
microscopical  examination,  reveal  the  fact  that  the  B.  anthracis,  during 
its  sojourn  in  the  body  of  the  insect,  degenerates.  Finally,  the  doctor 
states  that  it  is  conceivable  to  have  an  inoculation  in  an  abrased  skin 
surface  by  violently  crushing  the  surfeited  bloodsucker  against  the 
wound. 

Bacteriological  Diagnosis.  —  Remove  some  of  the  effusion 
from  the  deeper  portions  of  the  suspected  pustule,  and  insti- 
tute plate  cultures  (for  special  methods  see  Technique,  page 
58),  and  if  typical  colonies  develop,  then  pure  cultures  are 
made,  and  animals  inoculated.  In  cases  of  suspected  abdominal 
anthrax,  the  faeces  and  vomit  must  be  examined.  In  cases  of 
lung  anthrax  the  bacillus  is  sometimes  found  in  the  copious  expectora- 
tion. Examination  of  the  blood  reveals  whether  general  infection  exists 
or  not,  and  is  of  great  account  regarding  the  prognosis  of  the  case,  but 
it  must  also  be  remembered  that  the  bacilli  are  principally  localized  in 
the  capillaries. 

In  animals  it  is  often  necessary  to  decide  if  an  animal  died  of 
anthrax  or  not.  If  shortly  after  death  a  microscopical  examination  is 
made  of  the  blood  from  a  foot,  ear,  tail,  or  the  spleen  (but  it  is  considered 
advisable  never  to  cut  open  a  suspected  anthrax  carcase,  as  it  only 
favours  the  development  of  spores,  which  are  never  formed  in  the 


FIG.  37. — B.  Anthracis,  showing  commencing  involution  forms  and 
five  spores.     Fuehsin.     X  1000.' 


FHS.  8S.— B.  (Edematis  Maligni. 
Stab  culture  in  gelatine. 


Fio.  3D.— B.  CEdematis  Maligni.    Cover-glass  specimen  from  inoculated 
mouse.     Cladius  stain.     X  1000. 


[T.  Boa-hill,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


MALIGNANT  (EDEMA  113 

living  animals  or  unopened  carcases),  and  bacilli  are  detected  exhibiting 
capsules  when  stained  by  Johne's  method  (see  Technique,  §  22), 
then  the  diagnosis  is  assured. 

When  an  animal  has  been  dead  for  several  days,  bacilli  develop  in 
the  cadaver  somewhat  difficult  to  differentiate  from  the  Bacillus 
anthracis.  In  such  cases  gelatine  plate  cultures  must  be  instituted,  and 
mice  and  guinea-pigs  inoculated.  In  the  inoculation  of  animals,  the 
presence  of  the  bacillus  of  malignant  oedema  must  be  guarded  against, 
as  it  is  often  present,  the  inoculated  animal  dying  of  malignant  oedema 
in  spite  of  the  presence  of  the  Bacillus  anthracis.  This  error  can  be 
prevented  if  the  animals  are  '  inoculated  cutaneously  '  with  the  suspected 
material,  when  only  the  Bacillus  anthracis  develops,  and  a  false  diagnosis 
is  avoided.  When  putrefaction  is  too  far  advanced  in  a  carcase  under 
examination,  the  anthrax  bacilli  are  sometimes  completely  annihilated 
by  the  concurrence  of  other  species,  so  that  a  diagnosis  is  no  longer 
possible. 


MALIGNANT    (EDEMA. 

(Ger.  Bacillus  (Edematis  Maligni.     Fr.  Vibrion  Septique.) 

This  organism  is  very  widely  distributed  in  nature ;  it  is  found  in 
manured  garden  earth,  in  filth  and  dust,  in  house  drains,  and  also  in 
the  intestines  of  animals. 

Microscopical  Appearances. — The  bacilli  are  somewhat  narrower 
than  the  Bacillus  anthracis,  and  about  the  same  length,  but  differ  in  the 
ends  of  the  rods  being  rounded,  and  not  squarely  cut  across.  The 
peculiar  bamboo  rod  shape  found  in  anthrax  bacilli  is  also  absent. 

Motility. — Motile,  possessing  3  to  12  flagella,  which  are  attached  at 
the  ends  as  well  as  the  sides  of  the  rods  (see  Photomicrograph,  Plate  I., 
Fig.  5)  ;  the  motility  is  somewhat  weak.  For  special  methods  of  staining 
flagella,  see  Technique,  §§  23,  24. 

Staining  Reactions. — The  bacilli  obtained  from  the  animal  body, 
and  from  young  cultures,  stain  easily  with  the  ordinary  aniline  dyes. 

By  the  Gram  method  positive  results  are  only  obtained  when  the 
specimen  is  stained  for  twenty-four  hours  at  37°,  or  with  equal  parts  of 
anilin  water,  alcohol,  and  5  per  cent,  carbolic  acid  solution  of  gentian 
violet  for  fifteen  minutes.  With  the  Cladius  method  the  bacilli  are 
easily  stained  in  a  few  minutes. 

Spore  Formation. — The  spores  are  formed  generally  in  the  middle 
of  the  rods,  and  are  sometimes  broader  than  the  bacillus,  forming  a 
spindle  or  clostridium. 

Biological  Characters. — The  bacillus  is  strongly  anaerobic,  grow- 

H 


114  SPECIAL  BACTERIOLOGY 

ing  in  the  ordinary  media  at  room  and  incubator  temperature,  but  only 
in  the  absence  of  oxygen. 

On  Gelatine  Plates  the  growth  is  similar  to  the  Bacillus  subtilis. 

On  Agar  Plates  it  forms  a  thick  network  of  threads. 

In  Stab  Cultures  in  grape  sugar  gelatine  the  growth  begins  about  J  to 
J  of  an  inch  below  the  surface  of  the  medium  in  the  form  of  a  white  strip, 
with  side  branches  sprouting  out  (see  Photograph,  Fig.  38). 

On  Agar  Stab  Cultures  the  growth  exhibits  nothing  characteristic. 

Bouillon  becomes  cloudy  from  the  formation  of  gas. 

Growing  cultures  have  a  most  unpleasant  smell. 

In  Milk  a  part  of  the  casein  is  precipitated. 

Pathogenesis. — Affects  guinea-pigs,  rabbits,  and  mice.  The  strength 
of  pure  cultures  when  inoculated  into  susceptible  animals  is  very  variable. 
When  a  small  pocket  is  made  in  the  skin  of  a  mouse  and  as  much 
malignant  oedema  containing  earth  as  will  cover  the  point  of  a  knife 
introduced,  the  animal  will  die  in  one  to  two  days.  Examination  shows 
a  general  subcutaneous  oedema,  which  contains  large  numbers  of  the 
bacilli  (see  Photomicrograph,  Fig.  39).  The  bacilli  do  not  pass  into  the 
internal  organs  unless  the  animal  is  left  lying  some  time  after  it  is  dead. 

Two  cases  of  malignant  oedema  are  recorded  in  man.  They  were  sick 
with  typhoid  fever,  and  died  three  days  after  receiving  a  musk  injection, 
by  which  means  some  malignant  oedema  colonies  were  introduced  into 
the  subcutaneous  tissue.  The  infection  of  a  healthy  man  has  not  been 
observed.  Koch  mentions  a  case  of  mixed  infection  of  malignant 
oedema  and  anthrax  in  a  guinea-pig.  The  following  other  animals  are 
also  susceptible  : — Goats,  calves,  sheep,  horses,  swine,  cats,  dogs,  chickens, 
pigeons,  and  ducks  ;  while  cattle  are  immune. 

The  virus  is  weakened  when  passed  through  the  body  of  a  white  rat. 

Immunity.  —  Chamberland  and  Roux  rendered  guinea-pigs 
immune  by  injecting  intraperitoneally  bouillon  cultures  which  had 
been  sterilized  in  an  autoclave  ten  minutes  at  105°  to  110°  C. 


BACILLUS    ANTHRACIS    SYMPTOMATIC! 

(Ger.  Bact.  des  Rauschbrands ;  Fr,  Bact.  du  Charbon  Sympto- 
matique ;  Eng.  Symptomatic  Anthrax,  Black  Quarter,  etc.) 

This  organism  was  first  discovered  by  Bollinger  in  cattle  affected 
with  symptomatic  anthrax,  and  further  studied  by  MM.  Arloing, 
Cornevin,  and  Thomas, — Kitasato  being  the  first  to  obtain  cultures 
on  solid  media. 

The  characteristic  lesions  produced  by  this  organism  are  emphy- 
sematous  swellings  of  the  muscular  and  subcutaneous  tissues  of  the 


BACILLUS  ANTHRACIS  SYMPTOM ATICI  115 

leg  and  the  quarter,  accompanied  with  the  formation  of  gases  with  a 
strong  odour,  the  following  analysis  being  given  by  Kitt :  Co2  13  per 
cent.,  H.  76  per  cent.,  N.  10  per  cent.  On  section  of  the  affected 
parts  the  muscles  and  cellular  tissue  are  found  saturated  with  bloody 
serum,  while  the  muscular  tissue  is  dark,  almost  black,  in  colour.  In 
the  affected  areas,  in  the  gall,  and  after  death  in  the  internal  organs, 
the  above  organism  can  always  be  detected.  It  is  also  found  in  the 
soil  in  some  localities. 

Microscopical  Appearances. — The  bacilli  are  actively  motile  rods, 
3  to  5  /x  long,  and  0'5  to  0'6  ^  thick,  with  rounded  ends,  usually 
occurring  singly,  but  sometimes  forming  short  threads.  The  flagella  are 
attached  around  the  periphery  of  the  organism,  peritricha  (see  Photo- 
micrograph, Plate  I.,  Fig.  4).  The  bacillus  forms  spores,  situated  either 
in  the  middle  or  at  the  end  of  the  rods  (see  Photomicrograph,  Fig.  41). 
The  organism  commences  to  grow  at  16°  to  18°  C.,  but  spore  formation 
takes  place  best  at  37°  C.,  and  during  this  stage  the  organism  becomes 
motionless,  being  only  motile  during  the  vegetative  stage.  The  organism 
also  undergoes  degenerative  changes,  and  involution  forms  are  often 
present,  not  only  in  cultures  but  in  the  tissues  of  affected  animals. 

Staining  Reactions. — This  organism  stains  by  the  ordinary  aniline 
dyes,  and  by  the  Gram  method  only  when  the  staining  process  is 
prolonged.  It  stains  easily  by  the  Cladius  method.  The  spores  can 
also  be  stained  by  the  ordinary  methods  (see  Technique,  p.  27). 

Biological  Characters.  —  The  organism  is .  strictly  anaerobic, 
growing  best  in  an  atmosphere  of  hydogen,  but  not  in  carbon  dioxide. 

On  Gelatine  Plates  in  an  atmosphere  of  hydrogen  the  colonies  appear 
as  irregular,  slightly  lobulated  masses.  The  gelatine  liquefies  in  a  short 
time,  the  colony  then  presenting  a  dark  lobulated  centre  surrounded  by 
a  delicate  fringed-like  zone. 

In  Gelatine  Stab  Cultures  a  radiating  cloudiness  appears,  which  increases 
in  size  as  the  gelatine  softens,  until  finally  the  growth  resembles  a 
caterpillar  (see  Photograph  of  Culture,  Fig.  40),  where  the  characteristics 
of  the  growth  correspond  to  those  described  by  Sanfelice. 

In  deep  stab  cultures  in  grape  sugar  agar-agar  at  37°  C.  the  growth 
commences  in  twenty-four  to  forty-eight  hours,  accompanied  by  gas 
formation,  which  is  considerably  more  than  is  produced  by  the  bacillus 
of  malignant  oedema.  The  gaseous  products  have  a  putrid  odour,  like 
that  produced  by  rancid  butter. 

Milk  is  coagulated  by  the  formation  of  an  acid. 

Differential  Diagnosis. — This  organism  is  distinguished  from  the 
bacillus  of  malignant  oedema  as  follows : — It  is  smaller  and  does  not 
develop  in  long  threads  in  the  animal  tissues,  is  more  actively  motile, 
and  forms  spores  more  readily  in  the  living  tissues  than  does  the  bacillus 


116  SPECIAL  BACTERIOLOGY 

of  malignant  oedema.  It  also  differs  in  its  reaction  towards  animals. 
Cattle,  while  susceptible  to  symptomatic  anthrax,  are  practically  immune 
to  malignant  oedema.  Swine,  dogs,  rabbits,  pigeons,  and  chickens  are 
readily  affected  by  malignant  oedema,  but  not  as  a  rule  by  symptomatic 
anthrax.  According  to  Arloing,  frogs  can  be  inoculated  if  kept  at  a 
temperature  of  22°  C.  Horses  are  affected  only  locally  and  not  seriously 
with  symptomatic  anthrax,  but  are  conspicuously  susceptible  to  both 
artificial  inoculation  and  material  infection  by  the  bacillus  of  malignant 
oedema.  The  distribution  of  the  organisms  over  the  earth's  surface  is 
also  quite  different,  malignant  oedema  being  present  in  almost  all 
soils,  while  symptomatic  anthrax  appears  confined  to  certain  localities, 
especially  places  where  infected  herds  have  been  pastured.  Natural 
infection  occurs  principally  in  young  cattle ;  next  in  order  being  sheep 
and  goats  ;  whilst  in  mankind  a  genuine  case  is  not  recorded. 

The  ordinary  manner  of  infection  is  by  wounds  which  not  only  tear 
the  skin,  but  penetrate  the  subcutaneous  tissue.  The  disease  is  also 
produced  by  the  ingestion  of  forage  soiled  by  very  active  virulent 
matters,  and  by  the  inhalation  of  dust  charged  with  dried  virus. 

Vaccination. — Animals  sometimes  inoculate  themselves  accident- 
ally, and  as  small  doses  cause  immunity — this  immunity  being  trans- 
mitted from  the  mother  to  the  foetus — the  result  is  that  part  of 
the  animals  exposed  to  contagion  escape  its  fatal  effects.  The 
French  recommend  the  successive  employment  of  two  vaccines  pre- 
pared from  material  from  a  fresh  lesion  spread  out  thinly,  and  dried 
at  35°  C.,  and  then  a  sufficient  quantity  of  this  powder  is  triturated 
in  a  mortar  with  2  parts  of  water,  and  exposed  to  100°  to  104°  C. 
for  the  first  vaccine,  and  90°  to  94°  C.  for  the  second,  during  seven 
hours ;  when  the  dry  vaccines  are  taken  from  the  oven  1  centigram 
of  the  powder  is  diluted  in  J  c.c.  of  water  for  each  animal.  The 
vaccine  prepared  at  100°  is  used  first,  and  the  second  in  eight  days. 
The  inoculation  is  made  in  the  cellular  tissue  of  the  ear,  or  on  the 
internal  face  of  the  end  of  the  tail,  the  second  inoculation  a  little 
above  the  first.  Kitt  recommends  a  single  vaccine  from  infected  flesh 
heated  six  hours  at  100°  C.,  given  in  decigram  doses,  injected  in  the 
subcutaneous  tissue  near  the  elbow.  Animals  can  also  be  vaccinated 
with  natural  virus  either  in  the  cellular  tissue  or  intravenously ;  when 
the  latter  method  is  adopted  great  care  must  be  taken  to  avoid 
inoculation  of  the  surrounding  tissue.  Attenuated  viruses  are  there- 
fore generally  preferred  for  the  production  of  immunity. 

TETANUS. 

(FMg.  Lockjaw;  Ger.  Wundstarrkrampf ;  Fr.  Tetanos.) 
This  disease  occurs  in  all  the  domestic  animals  and  in  man.     The 


FIG.  40. —  B.  Antliracis 
Symptomatic!.  Stab  cul- 
ture in  grape  sugar  gelatine. 
(Caterpillar  form.) 


;.  41.— B.  Antliracis  Symptomatici  and  .spores.    Cover-glass  specimen 
from  grape  sugar  agar  culture.     Cladius  stain.     X  1000. 


^f         €**• 
^*"*»      *m  #      <yr 


%/r?, 
€t^,^    ^ 


FI(;.  4^.— B.  Tetani.    Stab  cul-         Fl°-  43.— B.  Tetani  and  spores.    Cover-glass  specimen  from  grape 
ture  in  grape  sugar  agar.    (Pine  sugar  agar  culture.     Fuchsin.     x  1300. 

tree  growth.) 

IT.  Jiov'MU,  F.n.C'.V.S.}  Phntn.,  Edinburgh,  1808, 


TETANUS  117 

horse,  ox,  sheep,  and  goat  are  the  most  susceptible,  and  it  has  also 
been  observed  in  the  pig  and  dog.  Chickens  are  immune.  Nicolier 
produced  tetanus  in  mice  and  rabbits  by  subcutaneous  inoculation 
with  particles  of  garden  earth  in  1884 ;  and  Kitasato  cultivated  the 
bacillus  of  tetanus  in  pure  cultures  in  1889. 

Microscopical  Appearances. — The  tetanus  bacillus  is  a  slender  rod 
with  rounded  ends  3  to  5  ft  long,  and  0*3  to  0'5  //,  wide,  and  may  appear 
as  single  rods,  or  in  cultures  as  long  threads. 

Motility. — Motile,  although  not  actively  so ;  the  flagella  are  attached 
somewhat  similarly  to  those  of  the  bacillus  of  malignant  oedema  (see 
Photomicrograph,  Plate  I.,  Fig.  6). 

Staining  Reactions. — It  stains  with  the  ordinary  aniline  reagents, 
and  by  the  Gram  and  Cladius  methods. 

Spore  Formation. — At  37°  C.  spores  are  formed  in  thirty  hours, 
and  at  room  temperature  in  about  a  week.  The  spores  are  situated  at 
one  end  of  the  rod,  and  have  a  diameter  of  1  to  1'5  /A,  giving  the  rod 
the  appearance  of  a  drumstick  (see  Photomicrograph,  Fig.  43,  x  1300). 

Excluded  from  air  and  light,  the  spores  in  a  culture  remain  living 
and  virulent  for  over  a  year.  They  can  also  resist  heating  to  80°  C.,  while 
exposure  to  steam  at  100°  C.  kills  them  in  five  to  eight  minutes.  They 
resist  the  action  of  5  per  cent,  carbolic  acid  tor  ten  hours,  but  succumb 
when  exposed  for  fifteen  hours ;  but  if  0-5  per  cent,  hydrochloric  acid  is 
added  they  are  no  longer  active  in  two  hours.  Corrosive  sublimate  1  to 
1000  kills  them  in  three  hours,  and  when  0'5  per  cent,  hydrochloric 
acid  is  added  they  are  killed  in  thirty  minutes. 

Biological  Characters. — The  tetanus  bacillus  is  anaerobic.  Recent 
observations,  however,  point  to  the  possibility  of  it  having  an  aerobic 
existence  (Flugge).  It  grows  well  in  an  atmosphere  of  hydrogen,  but 
not  in  carbonic  acid. 

Kitasato  was  the  first  to  isolate  the  bacillus  in  pure  cultures,  and  the 
following  is  the  method  he  adopted. 

Method. — Inoculate  several  white  mice  from  the  secretions  from  a 
wound  in  a  typical  case  of  tetanus.  The  material  usually  contains  other 
organisms  besides  tetanus  bacilli,  causing  more  or  less  suppuration  at  the 
seat  of  inoculation  in  the  mice. 

To  separate  the  tetanus  bacillus  from  others  present,  smear  the  pus 
upon  several  oblique  serum  and  agar-agar  tubes,  and  place  at  37°  to  38°  C. 
After  twenty-four  hours  all  the  organisms  will  have  developed,  and 
microscopic  examination  will  reveal  the  presence  of  a  few  tetanus  bacilli, 
recognisable  by  their  shape,  like  a  small  pin,  the  spore  representing  the 
head.  After  the  culture  has  remained  forty-eight  hours  at  38°  C.,  it  is 
subjected  to  a  temperature  of  80°  C.  in  a  water-bath  for  from  three- 


118  SPECIAL  BACTERIOLOGY 

quarters  to  one  hour.  A  series  of  cultures  are  now  instituted  in  media 
specially  prepared  for  the  growth  of  anaerobic  organisms  by  the  addition 
of  1-5  to  2'0  per  cent,  of  glucose. 

Kitt  obtained  pure  cultures  without  using  the  heating  process. 

The  original  material  was  reduced  with  sterile  water,  and  the  liquid 
inoculated  by  means  of  stroke  cultures  on  the  surface  of  horse  or  sheep 
serum  media,  and  the  cultures  placed  in  an  atmosphere  deprived  of  its 
oxygen  by  Buchner's  method  (see  page  66). 

On  Gelatine  the  colonies  grow  slowly,  the  central  portion  being  of  a 
golden  yellow  colour,  with  numerous  threads  radiating  from  the  centre. 

On  Agar-Agar  the  colonies  are  very  characteristic,  the  naked-eye 
appearance  being  that  of  fine,  fleecy  clouds,  which  under  the  microscope 
resemble  a  tangled  mass  of  fine  threads.  The  extraordinary  fineness  of 
the  latter  enables  the  colonies  to  be  distinguished  from  other  anaerobic 
organisms. 

In  Gelatine  Stab  Cultures  the  growth  has  the  appearance  of  a  cloudy, 
linear  mass  with  outgrowths  radiating  into  the  gelatine  from  all  sides  ; 
liquefaction  follows  slowly,  with  a  coincident  production  of  gas  with  an 
unpleasant  empyreumatic  smell. 

In  Agar  Stab  Cultures  the  growth  has  a  very  characteristic  appearance, 
resembling  a  fir-tree  (see  Photograph,  Fig.  42). 

Bouillon  is  densely  clouded. 

In  Milk  the  bacilli  grow  without  causing  any  changes. 

On  Potatoes  a  moist  invisible  growth  similar  to  that  of  the  Bacillus 
typhi  abdominalis  occurs. 

Pathogenesis. — Mice  inoculated  with  a  minute  portion  of  a  pure 
culture  of  the  tetanus  bacillus  develop  tetanic  symptoms  in  twenty-four 
hours,  which  end  fatally  in  from  two  to  three  days.  Rats,  guinea-pigs, 
and  rabbits  are  similarly  affected,  but  require  larger  doses  than  mice.  A 
fatal  dose  for  a  rabbit  is  0*3  to  0*5  c.c.  of  a  well-developed  bouillon  culture. 
The  period  of  incubation  for  rats  and  guinea-pigs  is  twenty-four  to 
thirty  hours,  and  for  rabbits  two  to  three  days.  Pigeons  are  but  slightly 
if  at  all  susceptible. 

The  tetanic  convulsions  appear  first  in  the  neighbourhood  of  the 
point  of  inoculation,  finally  becoming  generalized. 

At  the  autopsy  there  is  usually  only  a  small  hsemorrhagic  spot  at  the 
point  of  inoculation.  No  other  changes  are  present,  and  the  bacilli  are 
only  found  at  the  point  of  inoculation  (see  Photomicrograph,  Fig.  44), 
although,  according  to  Schnitzler,  they  are  sometimes  found  in  the 
lymph-glands  in  direct  relation  with  the  inoculated  part.  Buedinger 
found  that  tetanus  could  be  conveyed  from  an  animal  dead  of  it  to  a 
healthy  animal  by  transplanting  from  one  to  the  other  portions  of  the 
lymph-glands  associated  with  the  seat  of  infection. 

Death  results  from  the  absorption  of  a  soluble  poison  which  has  been 


TETANUS  119 

isolated  and  studied ;  possessing,  according  to  Kitasato,  the  following 
peculiarities.  When  cultures  of  the  tetanus  bacillus  are  filtered  through 
porcelain,  the  filtrate  contains  the  soluble  poison,  which  when  injected 
into  animals  causes  tetanus.  Animals  inoculated  with  pieces  of  the 
organs  of  animals  dead  from  the  action  of  the  tetanus  poison  are 
unaffected ;  but  inoculation  with  the  blood  or  pleural  exudates  produces 
positive  results.  The  poison  is  therefore  largely  present  in  the  circulating 
fluids. 

Vitality. — The  greatest  amount  of  poison  is  produced  in  fresh, 
neutral  bouillon  very  slightly  alkaline  in  reaction.  The  poison  loses  its 
activity  when  exposed  for  Ij  hours  to  55°  C.,  twenty  minutes  to  60°  C., 
and  five  minutes  to  65°  C.  When  dried  at  the  temperature  of  the  body 
with  access  of  air,  the  poison  is  destroyed ;  but  dried  at  ordinary  room 
temperature,  or  at  a  similar  temperature  in  the  desiccator  over  sulphuric 
acid,  it  is  not  destroyed.  Diffuse  daylight  diminishes  the  virulence  of 
the  poison.  Its  intensity  is  preserved  for  a  much  longer  time  when 
kept  in  the  dark. 

Direct  sunlight  destroys  its  poisonous  properties  in  from  fifteen  to 
eighteen  hours. 

When  diluted  with  a  fixed  amount  of  water  or  bouillon  its  activity 
is  not  diminished. 

Mineral  acids  and  strong  alkalies  lessen  its  intensity. 
In  man  tetanus  is  a  toxic  disease  the  same  as  in  animals,  the  bacilli 
never  being  found  in  the  blood  or  organs,  but  localised  at  the  point  of 
inoculation,  and  lymph-glands  associated  with  the  seat  of  infection. 
The  period  of  incubation  ranges  between  one  and  twenty-two  days ;  in 
a  case  of  wound  infection  at  a  laboratory  the  incubation  period  was  four 
days.  The  shorter  the  time  between  the  infection  and  the  appearance 
of  tetanus,  the  more  pronounced  is  the  course  of  the  disease,  and  the 
worse  the  prognosis. 

In  cases  where  the  incubation  period  of  the  disease  was  one  to  ten 
days,  only  about  3  per  cent,  recovered.  When  the  period  of  incubation 
was  ten  to  twenty-two  days  25  per  cent,  recovered,  and  by  longer  periods 
of  incubation  as  high  as  50  per  cent,  recovered. 

Immunity  and  Cure  of  Tetanus  -with  Antitetanic  Serum  in 
Animals. — Behring  produced  immunity  in  mice  with  bouillon  cultures 
of  tetanus  bacilli  weakened  by  adding  trichloride  of  iodine.  Fedorf 
also  prepared  a  tetanus  antitoxin  in  a  dry  form  from  blood  serum. 
Antitetanic  serum  can  also  be  obtained  from  horses  immunised 
against  tetanus  in  a  similar  manner  to  that  employed  in  the  produc- 
tion of  diphtheria  antitoxin.  The  animal  selected  is  a  young  horse 
in  good  condition,  which  is  first  tested  with  mallein  and  then  with 
tuberculin  to  ensure  its  freedom  from  glanders  and  tuberculosis. 


120  SPECIAL  BACTERIOLOGY 

'  The  tetanus  bacillus  is  grown  in  a  bouillon  culture  for  four  or 
five  weeks,  filtered  through  porcelain,  and  a  small  quantity  of  the 
filtrate,  about  half  a  drop,  injected  into  the  horse  subcutaneously ; 
the  dose  is  again  repeated  in  three  days,  and  if  no  signs  of 
tetanus  appear,  in  about  three  days  1  drop  is  again  injected,  and 
the  process  repeated  for  several  months  with  continually  increasing 
doses  of  the  filtrate,  until  the  horse's  system  becomes  so  resistant 
to  the  toxine,  that  large  quantities  can  be  injected  without  ill 
effects.  Six  months  after  the  first  injection,  the  horse  will  prob- 
ably remain  unaffected  by  a  dose  of  2  ounces,  and  at  the  end  of 
twelve  or  eighteen  months  as  much  as  a  pint  can  be  injected  with 
impunity.  At  frequent  intervals  during  this  process,  small  quantities 
of  blood  are  removed  from  the  horse,  and  the  potency  of  the  serum 
tested  experimentally  on  mice  and  guinea-pigs.  When  it  is  found 
to  confer  sufficient  immunity,  the  horse  is  periodically  bled  by  piercing 
the  jugular  vein  with  a  trocar  connected  by  sterilized  tubing  with  a 
sterilized  and  sealed  glass  flask.  The  blood  obtained  is  allowed  to 
coagulate,  and  its  serum  is  separated  and  filtered  for  use.  When  the 
blood  of  the  horse  once  acquires  a  sufficient  protective  power  to  be 
serviceable,  the  animal  will  continue  to  supply  potent  serum  for  a 
long  and  varying  period  without  further  dosing ;  but  the  supply 
must  be  continually  tested  experimentally.'' — (Sidney  Villar,  F.R.C.V.S., 
Proceedings  of  Royal  Counties  Veterinary  Association,  Nov.  1897). 

There  are  other  two  preparations  in  the  market  at  present,  one 
being  in  a  powder  form  which  is  dissolved  in  distilled  water  by  heat 
not  to  exceed  40°  C.,  and  as  a  curative  agent  in  horses  it  is  injected 
intravenously,  as  it  is  claimed  that  injection  direct  into  the  circula- 
tion yields  results  twenty-four  hours  quicker  than  subcutaneously. 
The  second  preparation  is  delivered  in  solution,  and  is  used  as  a 
prophylactic  in  man  and  animals  to  counteract  tetanus  in  wounds, 
in  which  experience  leads  one  to  expect  the  disease  to  develop.  The 
amount  of  the  dose  (0'5  to  5  c.c.)  is  regulated  by  the  time  that  has 
elapsed  since  the  wound  occurred.  In  operations  on  animals  often 
followed  by  tetanus,  e.g.y  before  castration,  docking,  etc.,  0'2  c.c.  is  a 
sufficient  dose. 

Nocard  considers  antitetanic  serum  valuable  as  a  prophylactic. 

In  some  parts  of  France  where  tetanus  is  very  prevalent,  Nocard 
distributed  antitetanic  serum  to  sixty-three  veterinary  surgeons,  who 
treated  for  the  prevention  of  tetanus  2727  animals  with  it.  Only  one 
animal  became  affected,  and  this  horse  was  not  inoculated  until  five 
days  after  being  pricked  in  shoeing.  Although  the  delay  was  too 
great  to  prevent  the  appearance  of  tetanus,  yet  the  disease  was  of  a 


CEREBRAL  TETANUS  121 

very  mild  nature.     During  the  same  period  these  veterinary  surgeons 
saw  259  cases  in  animals  that  were  not  so  inoculated. 

M'Watt  records  a  case  in  the  Brit.  Med.  Journal  of  a  boy  affected 
with  tetanus,  whose  recovery  he  attributed  to  the  use  of  Tizzoni's 
antitetanic  serum. 


CEREBRAL    TETANUS. 

At  the  Ninth  International  Congress  of  Hygiene,  held  at  Madrid, 
the  result  of  the  experiments  of  Dr  Borrel  and  Dr  Roux  regarding  the 
tetanic  disease  produced  by  the  direct  inoculation  of  tetanic  toxin  in  the 
brain  substance  of  susceptible  animals,  was  communicated  by  Dr  Borrel. 
The  special  malady — cerebral  tetanus — produced  is  defined  by  a  set  of 
symptoms,  including  excitement,  epileptiform  convulsions,  strange  or 
manifold  desires,  and  other  symptoms,  varying  according  to  the  portion 
of  the  brain  in  which  the  antitoxin  is  introduced.  This  tetanic  malady 
is  absolutely  different  from  the  ordinary  tetanus,  but  is  none  the  less 
specific.  Dr  Borrel  and  Dr  Roux  also  discovered  that  an  animal 
rendered  immune  against  injections  under  the  skin  was  not  immune 
against  injections  into  the  substance  of  the  brain,  and  from  this  they 
conclude  that  subcutaneous  injections  of  antitoxin  do  not  affect  the  nerve 
cells  and  do  not  protect  them.  The  same  occurs  when  for  therapeutic 
purposes  serum  is  injected  into  an  animal  which  has  commenced  to 
manifest  the  symptoms  of  tetanus,  the  nerve  substance  being  already 
attacked  by  the  toxin.  The  serum  does  not  reach  the  nerve  cells, 
and  therefore  the  toxin  can  continue  its  deadly  effects  undisturbed. 
This  explains  the  many  failures  to  treat  tetanus  by  antitoxin.  The  same 
authorities  then  tried  to  treat  the  disease  by  conveying  the  antitoxin 
direct  to  the  brain  substance,  and  were  thus  able  to  cure  rabbits,  guinea- 
pigs,  etc.,  even  when  the  disease  had  prevailed  for  some  hours,  and  this 
at  a  time  when  very  large  subcutaneous  injections  had  proved  of  no  use 
whatever.  On  the  other  hand,  when  injected  into  the  brain  matter 
very  small  quantities  of  serum  were  sufficient.  These  researches  on 
cerebral  tetanus  demonstrated  that  with  animals  immunized  passively  or 
actively  the  nerve  cells  are  not  immunized.  An  immunized  animal 
which  resists  a  subcutaneous  inoculation  of  the  toxin  will  die  if  the 
smallest  quantity  of  this  toxin  is  introduced  into  the  brain  matter. 
Immunity  is  not  therefore  due  to  some  new  property  acquired  by  the 
nerve  cells,  as  they  remain  unaffected  unless  directly  attacked. 


DIFFERENTIAL    DIAGNOSIS    TABLE 


122 


SPECIAL  BACTERIOLOGY 


DIFFERENTIAL    DIAGNOSIS    TABLE. 


ANTHRAX. 

MALIGNANT 

(EDEMA. 

TETANUS. 

SYMPTOMATIC 
ANTHRAX. 

(1)  Where  present 
in  the  animal 
tissues 

In  the  blood  and 
organs  in  large 
numbers 

Particularly  near 
the  point  of  in- 
fection and  in 
the  bloody 
oedema 

Scanty  in  the 
wound  secretion; 
never  in  the 
blood;  sometimes 
in  the  nearest 
lymph-glands  to 
the  point  of  in- 
fection 

In  the  bloody 
serous  exuda- 
tions at  the  point 
of  infection  and 
in  the  carcase 

(2)  Characteristic 
appearance  of 
the  organism 
in  the  tissues, 
blood,  etc. 

Rods  always  with- 
out spores;  cap- 
sules can  be  de- 
monstrated in 
cover  -  glass 
specimens 

In  single  rods  with 
rounded  ends, 
and  sometimes 
in  long  jointed 
threads 

Forms  spores  in  the 
body,  and  occurs 
in  rods  and 
threads  with  the 
peculiar  end 
spore  formation 
or  drumstick 

Forms  spores  in  the 
body,  which  are 
situated  either  at 
the  end  or  middle 
of  the  rod,  giving 
it  a  club  shape  ; 
sometimes  also 
forms  short 
threads 

(3)  Motility 

Non-motile 

Motile,  but  not 
always 

Motile,  principally 
vegetative  forms, 
without  spores 

Motile,  principally 
vegetative  forms, 
without  spores 

(4)  Growth 

Aerobic 

Anaerobic 

Anaerobic 

Anaerobic 

(5)  Stab  Cultures 

Central  growth, 
seldom  without 
hair-like  forma- 
tions 

Mostly  without 
hair-like  forma- 
tions, also  in 
isolated  ovoid 
zones 

Generally  like  a  fir 

tree 

Grows  in  gelatine 
with  a  formation 
like  a  caterpillar 

(6)  In  milk 

Coagulation 

Coagulated  slowly 

Coagulation  absent 

Coagulated  quickly 

(7)  Fermentation 

Negative 

Present 

Present 

Present 

(8)  Reaction  to- 
vrards  the 
Gram  stain 

Good 

Only  by  a  pro- 
longed exposure 
to  the  staining 
reagents 

Good 

By  prolonged  ex- 
posure to  the 
staining  reagents 

(9)  Pathogenic  for 
the  folloTving 
experimental 
animals 

Mice,  guinea-pigs, 
and  rabbits 

Mice,  guinea-pigs, 
and  rabbits 

Mice,  guinea-pigs, 
and  rabbits 

Not  for  rabbits 

Bacteria  associated  with  Meat-Poisoning. 
BOTULISMUS  (VAN  ERMENGEN). 

Notwithstanding  numerous  investigations  as  to  the  cause  of 
meat  poisoning,  the  question  still  remained  somewhat  obscure  until 
Van  Ermengen  discovered  a  specific  anaerobic  organism,  the  Bacillus 
botulinus,  during  an  epidemic  at  Ellezelles,  Belgium.  Lender  the 
term  '  meat-poisoning '  two  complex  set  of  symptoms,  with  different 
clinical  manifestations,  are  included,  which  are  differentiated  by  the 
prevailing  symptoms.  The  one  form,  described  as  gastro-intestinal, 
appears  as  a  cholera  nostras,  a  simple  or  haemorrhagic  gastro-enteritis, 


BOTULISMUS  123 

accompanied  with  fever,  albumirmria,  and  skin  eruptions  of  various 
form  and  intensity.  The  gastro-intestinal  symptoms  occur  after 
eating  tainted  meat,  or  meat  from  animals  slaughtered  while  suffering 
from  pyaemia,  septicaemia,  and  puerperal  fever.  The  organisms 
mostly  identified  as  the  cause  of  these  symptoms  belong  to  the  coli 
group,  and  more  rarely  in  some  cases  to  the  proteus  group  of 
organisms. 

The  second  form  of  symptoms  are  analogous  to  those  of  the 
so-called  sausage-poisoning,  with  pronounced  nervous  symptoms  of 
central  origin,  secretory  and  motor  disturbances,  suspension  of  salivary 
secretion,  dryness  and  redness  of  the  mouth  and  pharyngeal  mucous 
membrane,  difficulty  in  swallowing,  hoarseness,  mydriasis,  ptosis,  etc. 
This  form  is  now  known  as  botulismus,  and  appears  after  the  con- 
sumption of  certain  kinds  of  blood  and  liver  sausages.  It  is  also 
caused  by  using  decayed  salt  fish,  smoked  meat,  hams,  preserved 
meats,  etc. 

Microscopical  Appearances  of  the  Bacillus  botulinus. — 
Large  rods  4  to  6  ft  long,  0*9  to  1*2  /x  broad,  with  slightly  rounded 
ends.  The  formation  of  threads  was  seldom  observed,  but  involution 
forms  were  frequent.  They  are  rarely  found  in  the  blood  and 
organs  of  infected  animals,  being  mostly  situated  at  the  point  of 
inoculation. 

Motility. — Slightly  motile,  possessing  four  to  eight  flagella. 

Staining  Reactions. — The  staining  is  easy,  and  positive  results 
are  obtained  with  the  Gram  method  when  the  alcohol  is  not  allowed  to 
work  too  long  during  the  process  of  decolorizing. 

Spore  Formation. — Spores  are  formed  in  the  cultures,  and  in 
organical  structure  they  are  ovoid  in  shape,  and  situated  usually  in  the 
end,  very  seldom  in  the  centre  of  the  rod. 

Vitality. — They  are  destroyed  by  a  temperature  of  85°  C.  in 
fifteen  minutes,  and  at  80°  C.  in  an  hour.  Five  per  cent,  carbolic  acid 
destroys  them  in  less  than  twenty-four  hours.  Dried  spores  exposed 
to  diffuse  daylight  still  produce  cultivations  in  three  months. 

Biological  Characters.  —  The  Bacillus  botulinus  is  strictly 
anaerobic,  and  grows  best  between  20°  and  30°  C. ;  at  over  35°  C.  spores 
are  no  longer  formed,  the  growth  is  not  so  luxuriant,  and  involution 
forms  appear. 

The  culture  media  must  be  decidedly  alkaline,  and  the  addition  of 
2  per  cent,  grape  sugar  favours  their  growth. 

On  Gelatine  Plates,  in  four  to  six  days,  round,  transparent,  brownish- 
yellow  colonies  develop,  having  a  thick,  lustrous,  granular  appearance, 
and  surrounded  by  a  small  liquefied  area ;  later  the  margins  of  the 


124  SPECIAL  BACTERIOLOGY 

colony  become  irregular  and  radiating,  finally  giving  off  variously 
shaped  processes. 

In  Gelatine  Stab  Cultures  round  whitish  growths  occur  along  the 
course  of  the  needle,  from  which  processes  sometimes  extend  into  the 
surrounding  medium,  the  gelatine  is  liquefied,  and  gas  is  quickly 
formed. 

Grape  Sugar  Bouillon  is  densely  clouded. 

In  Milk  there  is  a  slight  growth,  without  any  alteration  of  the 
medium. 

On  Potatoes  there  is  no  growth.  The  cultures  give  off  a  smell  of 
butyric  acid ;  on  media  containing  sugar,  the  bacilli  also  form  butyric 
alcohol,  hydrogen,  carbon  dioxide,  and  methan. 

Pathogenesis. — Affects  guinea-pigs,  mice,  and  monkeys.  One  or 
two  drops  of  a  liquefied  gelatine  culture,  given  on  a  piece  of  bread  or  in 
milk,  is  sufficient  to  kill  the  animals.  Cats  can  be  fed  with  large  doses 
without  being  affected,  but  when  inoculated  with  large  doses  (5  to  10 
c.c.)  they  die  in  one  to  two  days,  and  with  small  doses  (1  to  2  c.c.) 
in  eight  to  twelve  days. 

The  incubation  period  is  about  thirty-six  hours,  when  the  animals 
become  depressed,  do  not  care  to  move,  refuse  their  food,  and  on  the 
third  day  appear  stupid,  the  eyes  almost  motionless,  and  the  pupils 
greatly  dilated.  The  enlargement  of  the  pupil  increases  to  an  enormous 
extent  in  the  next  few  days.  The  tongue  hangs  out  of  the  mouth,  and 
finally  the  animal  cannot  retract  it ;  the  faeces  and  urine  are  withheld, 
death  usually  occurring  from  paralysis  of  the  respiratory  and  circulatory 
organs.  Very  small  doses  of  the  bacilli  cause  marasmus,  the  cats  dying 
in  several  weeks  with  symptoms  of  paralysis  and  degeneration  of  the 
internal  organs.  Pigeons  receiving  1  to  2  c.c.  of  a  culture  exhibit 
first,  paralysis  of  the  wings,  and  finally  general  paralysis. 

The  pathological  anatomical  changes  usually  present  are,  a  more 
or  less  well-marked  hyperaemia  of  most  of  the  organs  ;  in  some  instances 
acute,  sometimes  interstitial,  and  sometimes  parenchymatous  hepatitis, 
with  fatty  degeneration,  and  desquamative  parenchymatous  nephritis, 
fatty  degeneration  of  the  heart  muscle,  and  also  of  the  muscle  of  the 
eye.  The  degenerative  changes  found  in  the  central  nervous  system 
are  of  especial  interest.  In  the  spinal  cord  the  changes  are  confined 
almost  entirely  to  the  grey  substance  of  the  cord  along  the  anterior 
cornu.  In  the  medulla  oblongata  the  ganglion  of  the  hypoglossal  nerve, 
the  dorsal  ganglion  of  the  vagus,  the  middle  small-celled  ganglion  of 
the  motores  oculorum,  and  of  other  cranial  nerves,  are  affected. 
Cultures  obtained  from  fresh  organs  of  animals  inoculated  intravenously 
do  not  exhibit  a  very  pronounced  growth,  but  if  the  organs  are 
previously  placed  in  the  incubator  at  30°  C.  for  twelve  to  twenty-four 
hours,  numerous  bacilli  can  be  isolated.  The  Bacillus  botulinus  does 


BACILLUS  ENTERITIDIS  125 

not  cause  a  genuine  infection,  but  an  intoxication,  and  can  be  placed  in 
the  same  category  as  tetanus  and  diphtheria.  The  toxin  can  be  pre- 
cipitated with  alcohol,  tannic  acid,  and  neutral  salts.  Other  organisms 
found  associated  with  the  Bacillus  botulinus  appear  neither  to  favour 
or  retard  its  poisonous  products. 

Bacteriological  Diagnosis.  —  In  one  of  Van  Ermengen's  cases 
the  spores  of  the  Bacillus  botulinus  were  found  in  a  ham,  mostly  in 
the  red  or  lean  part,  seldom  in  the  fat.  For  animal  experiments  take 
four  parts  of  the  suspected  ham  and  cut  it  into  small  pieces,  and  add 
five  parts  of  sterilized  water,  and  inject  a  minute  quantity  of  the 
infusion.  Van  Ermengen  was  able  to  isolate  the  Bacillus  botulinus 
out  of  the  spleen,  stomach,  and  intestinal  contents  of  a  man  dead  from 
meat-poisoning. 

Immunity. — Kempner  produced  immunity  in  animals  with  the 
nitrate  of  a  bouillon  culture.  The  serum  of  the  immunized  animals 
was  highly  antitoxic,  and  doses  of  1  to  5  c.c.  injected  three  to 
twenty -four  hours  after  a  guinea-pig  was  poisoned  with  the  Bacillus 
botulinus  resulted  in  the  recovery  of  the  affected  animals.  Kempner 
and  Pollack^  investigations  show  that  after  twenty  hours  the  intoxica- 
tion causes  changes  in  the  central  nervous  system,  which  again  becomes 
normal  when  the  serum  is  used. 

BACILLUS   ENTERITIDIS. 

This  bacillus  was  discovered  by  Gartner  in  1888  in  a  meat-poison- 
ing epidemic,  and  since  then  has  been  observed  in  a  number  of  similar 
cases. 

Microscopical  Appearances. — Small  rods.  Hanging-drop  speci- 
mens from  gelatine  cultures  show  a  difference  between  the  centre  and 
the  ends  or  polar  portions  of  the  rods,  the  former  appearing  to  consist 
of  a  less  refractive  substance.  This  peculiar  difference  appears  only 
to  exist  in  gelatine  cultures. 

Motility. — Motile,  possessing  2  to  5  long  flagella. 

Staining  Reactions. — When  stained  with  the  ordinary  aniline  dyes, 
the  middle  portion  of  the  rod  is  strongly  stained,  while  the  ends  or  polar 
portions  are  either  weakly  stained  or  entirely  uncoloured.  The  reaction 
with  the  Gram  method  is  negative. 

Biological  Characters. — It  grows  at  both  room  and  incubator 
temperature  ;  by  the  latter  method  the  growth  is  quicker. 

On  Gelatine  Plates  the  colonies  developing  on  the  surface  of  the 
medium  appear  as  thin  transparent  films.  The  gelatine  is  not  liquefied. 

In  Gelatine  Stab  Cultures  the  growth  extends  along  the  whole  length 


126  SPECIAL  BACTERIOLOGY 

of  the  middle  puncture,  a  film  developing  on  the  surface ;  old  cultures 
gi  ve  off  a  slight  odour. 

On  the  surface  of  Agar  at  37°  C.  a  copious  grey  transparent  coating 
o  ccurs,  and  a  slight  odour  is  given  off. 

On  Potatoes  at  37°  C.  in  two  days  a  somewhat  copious  greyish-white 
shiny  coating  develops. 

Bouillon  cultures  exhibit  strongly-marked  cloudiness  throughout  the 
medium. 

Grape  Sugar  Bouillon  exhibits  the  same  changes  as  occur  with  the 
Bacterium  coli  commune ;  the  medium  becomes  strongly  acid,  and  gas  is 
formed,  sometimes  CO2  and  sometimes  a  combustible  gas. 

In  Milk  Sugar  Bouillon  no  acid  reaction  occurs ;  the  fluid  remains 
alkaline,  but  sometimes,  although  not  always,  small  gas  bubbles  are  found 
consisting  of  CO2  or  a  combustible  gas. 

Milk  is  not  altered  in  chemical  reaction  nor  coagulated. 

Indol  is  not  formed  in  twenty-four  hours'  old  cultures  at  37°  C.,  and 
the  Nitroso-indol  reaction  is  absolutely  negative. 

The  Bacillus  enteritidis  grows  excellently  under  aerobic  conditions 
in  the  ordinary  nutrient  media,  but  only  under  anaerobic  conditions  in 
the  presence  of  grape  sugar. 

Pathogenesis. — Mice  and  guinea-pigs  are  very  susceptible  to 
subcutaneous  inoculation,  while  rabbits  are  less  susceptible  to  infection. 
Mice  die  in  one  to  three  days,  guinea-pigs  in  about  five  days,  and  the 
inoculated  bacteria  are  again  found  in  the  heart's  blood  and  organs  of 
these  animals.  Mice  die  in  five  to  eight  days  when  fed  with  food 
containing  the  bacteria,  and  the  specific  bacteria  are  found  in  their 
internal  organs ;  guinea-pigs  are  also  infected  by  way  of  the  digestive 
tract. 

Ill  man,  this  bacillus  causes  gastro-enteric  symptoms  which  may 
be  more  or  less  intense,  occasionally  causing  death,  specific  bacteria 
being  found  in  the  organs.  They  appear  to  be  introduced  into  the 
intestinal  canal  with  the  food ;  very  probably  such  meat  is  obtained 
from  animals  in  which  the  Bacillus  enteritidis  was  already  present 
during  life.  In  what  animal  diseases  this  bacillus  can  possibly 
originate  is  not  known. 

BACILLUS   MORBIFICANS  BOVIS. 

This  organism  was  found  by  Basenau  in  a  cow  affected  with 
puerperal  fever. 

Microscopical  Appearances. — Rods  about  the  same  size  as  the 
typhoid  bacillus,  0*3  to  0'4  p  wide,  and  1  to  1*2  //.  long,  sometimes 
arranged  in  pairs. 

Motility. — Strongly  motile. 


THE  PROTEUS  GROUP  OF  BACTERIA  127 

Spore  Formation  absent. 

Staining  Reactions. — Easily  stained  with  the  ordinary  agents,  but 
not  by  the  Gram  method. 

Biological  Characters. — The  developing  colonies  resemble  those  of 
the  Coli  commune,  but  are  more  granular. 

In  Stab  Cultures  and  on  Agar  media  greyish-white  tufts. 

On  Potatoes  a  moist  yellow  coating  which  never  becomes  brown. 

Bouillon  is  clouded,  a  film  forming  on  the  surface. 

Milk  is  not  coagulated. 

Grape  Sugar  is  slightly  fermented,  but  Cane  Sugar  remains  unchanged. 

Vitality. — Cultures  are  killed  when  heated  one  minute  at  70°  C. 

Pathogenesis. — Mice,  rats,  guinea-pigs,  and  rabbits  are  very 
susceptible  to  infection  by  subcutaneous  and  intraperitoneal  inocu- 
lation, also  by  feeding.  Dogs  and  cats  are  immune.  The  above 
bacillus  is  also  probably  pathogenic  for  man,  as  many  cases  of  sickness 
have  been  observed  to  follow  the  consumption  of  the  flesh  of  animals 
affected  with  puerperal  fever  (Ostertag). 

THE  PROTEUS  GROUP  OF  BACTERIA. 

This  group  of  organisms  discovered  by  Hauser  in  1885  consists  of 
three  species — 

1.  Proteus  Vulgaris. 

2.  „       Mirabilis. 

3.  „       Zenkeri. 

(1)  PROTEUS  VULGARIS. 

Microscopical  Appearances. — Small  rods  of  various  sizes,  generally 
occurring  in  pairs,  but  sometimes  arranged  in  filaments.  Involution 
forms  frequently  occur,  the  most  common  being  spherical  bodies  about 
1*6  /x  in  diameter. 

Motility. — Strongly  motile,  possessing  numerous  flagella  (see  Photo- 
micrograph, Plate  II.,  Fig.  12). 

Spore  Formation  is  absent,  and  the  bacilli  are  killed  by  five 
minutes'  exposure  to  a  temperature  of  55°  C. 

Staining  Reactions. — The  bacilli  are  easily  stained  with  fuchsin, 
not  so  easily  with  the  ordinary  watery  solutions  of  the  dyes.  By  the 
Gram  method  the  reaction  is  negative. 

Biological  Characters. — It  is  a  facultative  anaerobe,  and  at  ordinary 
room  and  incubator  temperature  the  growth  is  equally  luxuriant,  the 
optimum  temperature  being  20°  to  25°  C. 

On  Gelatine  Plates  small  round  yellowish  colonies  with  thick  centres 


128  SPECIAL  BACTERIOLOGY 

and  irregular  edges  develop  at  first,  from  which  brush-like  offshoots  are 
thrown  out.  Other  colonies  are  surrounded  by  a  zone  of  threads  which, 
partly  in  circular,  partly  in  irregular  twisted  forms,  surround  the  central 
opaque  mass.  The  gelatine  is  quickly  liquefied.  Straight  and  twisted 
offshoots,  which  frequently  become  detached  from  the  mother-stem,  grow 
into  the  surrounding  medium,  and  continue  moving  in  the  somewhat 
softened  gelatine.  This  condition  is  known  as  Swarming  Islands  (see 
Photomicrograph  of  same,  Fig.  45),  which  is  easily  observed  in  cultures 
on  5  to  6  per  cent,  gelatine.  Peculiar  figures  and  designs  also  occur, 
whereby  the  proteus  has  been  designated  the  Bacillus  Jtgurans. 

Stab  Cultures  in  gelatine  liquefy  very  quickly. 

On  oblique  surface  Agar  a  rapidly  extending  moist  grey  layer  is 
formed. 

On  Potatoes  a  dirty  greyish  coating  develops. 

Bouillon  becomes  uniformly  clouded.  Cultures  on  all  the  different 
media  give  off  an  abominable  smell. 

Pathogenesis. — When  a  considerable  quantity  of  a  proteus  culture 
(3  c.c.)  is  injected  intravenously  or  into  the  peritoneum  of  a  rabbit  or 
guinea-pig,  the  animals  die  of  acute  enteritis  and  peritonitis.  The  intra- 
venous injection  of  5  to  10  c.c.  of  a  bouillon  culture  in  dogs  causes  more 
typical  symptoms  and  lesions.  The  symptoms  produced  are  bloody 
vomiting  and  diarrhoea,  combined  with  severe  tenesmus  and  elevation  of 
the  temperature.  At  the  autopsy  an  intense  haemorrhagic  enteritis  is 
found.  The  blood  and  internal  organs  either  contain  none  or  very  few 
bacilli.  With  filtered  cultures  and  with  cultures  containing  the  remains 
of  dead  bacilli,  the  same  results  are  obtained.  The  proteus  is  also  fatal 
to  mice,  and  the  bacilli  can  again  be  cultivated  from  their  organs,  and 
the  more  often  the  bacillus  is  passed  through  mice  the  more  virulent  it 
becomes. 

The  proteus  bacteria  are  found  in  all  putrefactive  processes,  and  in 
the  intestinal  canal.  In  man  the  proteus  forms  a  mixed  infection  with 
the  ordinary  exciters  of  inflammation.  They  cause  the  ichorous,  putrid 
phlegmon  sometimes  observed  in  cases  of  cadaveric  infection.  The 
proteus,  further,  sometimes  penetrates  suppurating  wounds,  and  increasing 
these,  forms  toxic  products  which,  when  absorbed,  cause  the  so-called 
'  putrid  intoxication.'  According  to  H.  Jager,  certain  forms  of  icterus, 
accompanied  with  fever,  pain  in  the  muscles,  and  enlarged  liver  and 
spleen,  known  as  '  Weil's  disease,'  are  produced  by  the  proteus.  Jager 
was  able  to  cultivate  from  the  urine,  and  after  death,  from  the  organs  of 
individuals  dead  of  Weil's  disease,  a  fluorescent  proteus.  The  infection 
resulted  in  these  cases  from  bathing  in  river-water  which  was  con- 
taminated with  the  proteus.  An  outbreak  of  disease  due  to  the  proteus 
fluorescens  also  occurred  among  some  poultry  kept  on  the  banks  of  a 
small  stream. 


44. — B.  Tetani.     Cover-glass  specimen  from  point  of  inoculation. 
Cladius  stain.     X  1000. 


Fi<;.  •!.">.- -li.  Proteus  Yulgaris./  Contact  specimen  from  Fi,;.  4«>.*—  Bacillus  Mallei.     Section  of  glanders  nodule 

gelatine  plate  (swarming  islands).     Fuchsin.     X  1000.  from  horse,  showing  single  bacillus.     Stained  by 

L'ittier's  method.     X  1000. 

*  For  the  tissue  from  which  this  specimen  was  obtained,  1  am  indebted  to 
Win.  Hunting,  F.K.C.V.H.,  Editor  of  Tin-  H'/mm/ 


\T.  Jiowliill,  FJl.C.V.*.,  Photo.,  Edinburgh,  1898. 


THE  PROTEUS  GROUP  OF  BACTERIA  129 

E.  Levy  found  the  proteus  to  be  the  cause  of  a  haemorrhagic  gastro- 
enteritis which  appeared  in  seventeen  persons  after  partaking  of  decom- 
posed meat. 

According  to  Booker,  the  proteus  vulgaris  plays  an  important  part  in 
the  production  of  the  morbid  symptoms  characteristic  of  cholera  infan- 
tum.  It  was  found  in  the  alvine  discharges  of  affected  infants,  but  not 
in  those  of  healthy  infants.  The  prominent  symptoms  in  the  cases 
where  the  proteus  was  found  were  drowsiness,  stupor,  emaciation,  more 
or  less  collapse,  frequent  vomiting  and  purging,  with  watery  and 
generally  offensive  stools. 

Bacteriological  Diagnosis. — Plate  cultures  are  prepared  from 
the  pus,  from  the  ichorous  phlegmons,  and  also  from  the  urine 
obtained  under  sterile  precautions  from  the  patients  affected  with 
Weil's  disease. 

(2)  PROTEUS  MIRABILIS. 

Microscopical  Appearance. — Rods  of  various  lengths,  the 
smallest  being  about  0'6  /x  in  length. 

Spore  Formation  absent. 

Biological  Characters. — On  Gelatine  Plates  the  deep-lying  colonies 
exhibit  curiously  formed,  twisted  zooglrea  masses.  The  surface  colonies 
occasionally  form  the  swarming  islands  like  the  Proteus  vulgaris.  The 
gelatine  is  liquefied  slowly. 

(3)  PROTEUS  ZENKERI. 

Microscopical  Appearance.— Bacilli  0'4  /*  broad,  and  averaging 
1-6  {J.  long. 

Spore  Formation  absent. 

Biological  Characters. — It  occasionally  forms  swarming  islands,  like 
the  Proteus  mirabilis ;  but  the  gelatine  is  not  liquefied. 

BACILLUS   MALLEI. 

(The  Glanders  Bacillus ;  Ger.  Rotzbacillus ;  Fr.  Bac.  de  la  Morve.) 

This  organism  was  discovered  by  Loifler  and  Schutz  in  1882  in 
the  diseased  tissues  of  animals  affected  with  glanders.  It  was  isolated 
in  pure  cultures,  which,  when  inoculated  into  susceptible  animals, 
reproduced  the  disease  with  all  its  clinical  and  pathological  manifesta- 
tions. 

Microscopical  Appearances.— Short  rods,  2  to  3  p.  long,  0-2  to 
0-4  //,  thick,  with  rounded  or  slightly  pointed  ends,  generally  occurring 
in  single  rods,  sometimes  in  pairs,  and  very  seldom  in  long  filaments. 

I 


130  SPECIAL  BACTERIOLOGY 

Motility. — Non-motile. 

Staining  Reactions. — The  bacilli  stain  readily  in  cover-glass  speci- 
mens with  the  ordinary  basic  aniline  dyes  (see  Photomicrograph,  Plate 
III.,  Fig.  16).  The  staining  is  somewhat  irregular,  the  bacilli  presenting 
a  granular  aspect  owing  to  alternating  clear  and  uncoloured  spaces. 
(For  special  staining  methods,  see  Technique,  §  15.)  They  are  not 
stained  by  the  Gram  method. 

The  bacilli  are  always  present  in  diseased  tissues,  although  great 
difficulty  is  usually  experienced  in  demonstrating  them  by  staining 
methods.  When  properly  stained,  they  are  found  most  numerous  in  the 
centre  of  the  nodules,  becoming  gradually  less  so  towards  the  periphery . 
Their  usual  position  is  between  the  cells,  but  sometimes  they  almost  fill 
some  of  the  epithelial  cells.  They  are  always  present  in  the  nodules 
(see  Photomicrograph,  Fig.  46),  rarely  in  the  blood,  and  if  so,  only  in 
small  numbers.  (For  special  methods  of  staining  the  bacilli  in  sections 
of  tissues,  see  Technique,  §§  43,  44.) 

Spore  Formation. — It  is  still  an  open  question  if  the  glanders 
bacillus  forms  spores  or  not.  Some  observers  claim  that  spores  are 
present.  The  evidence  of  spore  formation  cannot  be  accepted  when  the 
organism  is  subjected  to  the  following  tests : — 

(1)  The  Bacillus  mallei  does  not  at  any  stage  of  its  growth  resist 

exposure  to  3  per  cent,  carbolic  acid  solution  longer  than  five 
minutes,  nor  to  1  to  5000  corrosive  sublimate  solution  for  more 
than  five  minutes. 

(2)  It  is  destroyed  in  ten  minutes  in  some  experiments,  and  in  five 

minutes  in  others,  when  exposed  to  a  temperature  of  55°  C. 

(3)  When  dried,  according  to  various  authorities,  it  loses  its  vitality 

in  from  thirty  to  forty  days. 

The  conditions  exhibited  above  are  directly  opposed  to  the  existence 
of  spores. 

Biological  Characters. — The  Bacillus  mallei  is  a  facultative 
organism,  growing  both  with  and  without  oxygen.  It  develops  on  the 
ordinary  nutrient  media.  Minimum  temperature,  25°  C. ;  optimum, 
37°  C.  ;  maximum,  42°  C.  The  best  growth  is  exhibited  on  5  per  cent, 
glycerine  agar  medium. 

On  Agar  Media,  with  or  without  the  addition  of  5  per  cent,  glycerine, 
it  forms  a  moist,  opaque,  glazed  layer,  devoid  of  special  characteristics. 
Sometimes  no  growth  occurs  on  ordinary  agar. 

On  Blood  Serum  it  forms  a  moist,  opaque  layer  of  yellowish  or  dirty- 
brownish  drops.  The  serum  is  not  liquefied. 

On  Potatoes  the  Bacillus  mallei  exhibits  a  very  characteristic  growth, 
which  is  somewhat  rapid,  and  in  twenty-four  to  thirty-six  hours  at  37°  C. 
a  moist,  amber-yellow,  transparent  deposit  appears,  becoming  deeper  in 


BACILLUS  MALLEI  131 

colour  and  denser  in  consistency,  until  it  finally  presents  a  reddish-brown 
colour  and  the  surrounding  surface  of  the  potato  becomes  darkened. 
According  to  Semner,  the  Bacillus  mallei  exhibits  unusual  pleomorphism 
on  potatoes,  often  forming  long  felt-like  interlaced  filaments,  not  unlike 
the  threads  of  the  Bacillus  anthracis,  and,  finally,  blebby  and  club-like 
swellings. 

In  Bouillon  a  diffuse  clouding  takes  place,  a  tenacious,  ropy  sediment 
being  ultimately  formed. 

In  Milk  Media  to  which  a  little  litmus  is  added,  the  blue  colour 
becomes  reddish  in  four  or  five  days,  and  quite  red  in  two  weeks  at 
37°  C.,  the  milk  being  finally  separated  into  a  firm  clot  of  casein  and 
clear  whey. 

Vitality. — Cultures  of  the  Bacillus  mallei  lose  their  virulence  very 
quickly  by  a  natural  weakening  as  early  as  the  fourth  and  fifth  genera- 
tions ;  therefore  to  retain  the  cultures  virulent  it  is  necessary  after  two 
or  three  culture  generations  to  pass  the  virus  through  a  susceptible 
animal.  According  to  Loffler  the  Bacillus  mallei  lives  three  months  in 
a  dry  condition,  while  other  authors  find  that  when  spread  out  in  a  thin 
layer  the  bacilli  die  in  ten  days.  When  exposed  to  heat  they  are  killed 
at  1 00°  C.  in  two  minutes,  and  at  80°  C.  in  five  minutes.  Exposed  to 
the  action  of  corrosive  sublimate  1  to  1000  they  are  killed  in  fifteen 
minutes,  and  in  5  per  cent,  carbolic  acid  in  one  hour ;  and  they  also 
lose  their  virulence  quickly  in  distilled  water  (six  days).  The  virulence 
is  not  destroyed  by  putrefaction ;  inoculations  made  with  central  por- 
tions of  glanders  lungs,  exposed  to  the  air  for  fifteen,  eighteen,  and 
twenty-six  days,  have  given  positive  results. 

Pathogenesis. — The  ass,  mule,  horse,  goat,  cat,  sheep,  dog,  pig,  and 
mankind,  are  susceptible.  Cattle  are  immune. 

Among  laboratory  animals,  the  field  mouse,  wood  mouse,  and  the 
guinea-pig  are  the  most  susceptible,  the  rabbit  being  less  so;  white 
mice  and  house  mice  are  immune.  Birds,  with  the  exception  of  the 
pigeon,  are  refractory. 

Field  mice  inoculated  subcutaneously  with  a  small  quantity  of  a  culture 
die  very  quickly — in  three  or  four  days.  The  spleen  is  found  enlarged 
and  generally  studded  with  minute  grey  nodules,  which  are  rarely  present 
in  the  lungs,  but  frequently  found  in  the  liver.  Pure  cultures  can  be 
obtained  from  these  nodules.  The  characteristic  lesions  are  much  more 
marked  in  the  guinea-pig,  which  lives  from  six  to  eight  weeks  after 
inoculation.  The  specific  inflammatory  condition  of  the  mucous  mem- 
brane of  the  nostrils  is  almost  always  present ;  the  joints  are  infiltrated 
and  swollen.  Orchitis  and  epididymitis  are  present  in  male  animals, 
while  the  internal  organs,  lungs,  kidneys,  spleen  and  liver  are  generally 
the  seat  of  the  characteristic  nodular  formations.  Pure  cultures  can  be 
obtained  from  the  diseased  centres. 


132  SPECIAL  BACTERIOLOGY 

Modes  of  Infection  and  Course  of  the  Disease. — According  to 
M.  Nocard,  the  only  means  of  experimentally  obtaining  miliary  tubercles 
in  the  lungs  of  horses  identical  with  those  found  in  cases  of  natural 
glanders,  is  to  cause  the  animal  to  ingest  virulent  matter,  cultures,  or  pus. 

Prieur,  according  to  Nocard,  gives  the  most  complete  and  most  lucid 
dissertation  of  the  actual  state  of  our  knowledge  of  the  farco-glanderous 
affection  (Veterinary  Itecord,  505,  1898). 

The  author  concludes  thus  : — 

'  1 .  The  glanders  virus  commonly  gains  entrance  into  solipeds  by 
means  of  the  alimentary  canal. 

'2.  Experimental  glanders,  determined  in  solipeds  by  the  glanders 
virus,  evolves  exactly  as  clinical  glanders. 

'  3.  Translucid  tubercles  are  of  a  glanderous  nature. 

'  4.  Certain  forms  of  cutaneous  glanders  may  in  man  be  cured  by 
the  means  of  very  energetic  and  rapidly  instituted  treatment. 

1 5.  Certain  cases  of  pulmonary  glanders  are  in  the  horse  capable  of 
cure  by  the  effect  of  the  sole  force  of  the  economy  aided  by  a  special 
hygiene. 

'  6.  The  difference  of  curability  in  man  and  the  horse  results  from  the 
ordinary  mode  of  contamination  of  each  species.  In  man  cutaneous 
glanders  is  an  incursive  glanders  (inoculation),  of  which  one  may  with 
success  endeavour  to  arrest  the  invading  march.  Cutaneous  glanders  in 
the  horse,  on  the  contrary,  is  a  recursive  glanders  infection,  the  manifes- 
tations of  which  indicate  defect  of  the  economy. 

( 7.  The  employment  of  mallein  is  the  sole  means  of  diagnosis  which 
we  have  at  our  disposal  in  those  cases  of  glanders  exempt  from  clinical 
signs. 

f  8.  An  animal  which  having  presented  a  complete  reaction  to  mallein 
and  does  not  react  again  after  a  variable  number  of  injections  of  the 
reagent,  may  be  considered  as  cured. 

'  9.  Rigid  application  of  the  rules  of  sanitary  police  remains  the  most 
efficacious  measure  of  opposing  the  development  of  glanders  in  man  and 
animals.' 

Schiiltz,  in  his  recent  work  on  the  Experimental  Pathology  and 
Pathological  Anatomy  of  Glanders,  formulates  the  following  conclusions 
(Veterinary  Record,  502,  1898): — 

'  1 .  Primary  pulmonary  glanders  is  not  developed  in  consequence  of 
the  introduction  of  the  specific  bacilli  into  the  alimentary  canal. 

'  2.  The  existence  of  primary  pulmonary  glanders  has  not  yet  been 
demonstrated. 

'  3.  The  grey  and  translucid  granulations  of  the  lungs  of  horses  are 
not  glanderous,  but  simply  inflammatory  lesions  provoked  by  a  parasite 
that  can  determine  similar  lesions  in  the  kidneys. 

'  4.  The  equine  pulmonary  glanderous  tubercle  is  a  nodule  hepatiza- 
tion  which  undergoes  a  special  disaggregation  (chromatotexis). 


BACTERIOLOGICAL  DIAGNOSIS  OF  GLANDERS         133 

'  5.  Old  glanderous  granulations  contain  giant-cells. 

'  6.  Glanderous  granulations  do  not  calcify,  whereas  parasitic  granu- 
lations do.' 

It  is  evident  from  the  above  extracts,  and  the  contradictory  nature  of 
the  same,  that  the  question  of  the  mode  of  the  entry  of  the  glanders 
virus  in  solipeds  is  still  open. 

In  man,  where  the  virus  enters,  a  local  swelling  appears,  which 
spreads  quickly,  accompanied  with  suppuration  and  cording  of  the 
neighbouring  lymphatics.  Multiple  abscesses  are  next  formed  in  the 
skin,  muscles,  and  internal  organs,  and  there  are  often  suppurative 
changes  in  the  joints  ;  at  this  stage  the  disease  resembles  pyaemia. 
Characteristic  glanders  nodules  appear  in  the  mucous  membranes, 
particularly  in  the  nose,  which  soon  disintegrate,  forming  ulcers.  Death 
is  caused  by  general  infection,  carried  by  means  of  the  lymph 
circulation. 

Heredity.  —  Loffler  observed  a  female  guinea-pig  which  resisted 
a  glanders  inoculation,  and  five  months  after  being  inoculated  gave 
birth  to  one  young  one,  which  at  birth  seemed  perfectly  healthy,  but 
died  in  a  week  from  glanders  of  the  viscera. 

BACTERIOLOGICAL   DIAGNOSIS    OF    GLANDERS- 
METHOD  OF  STRAUSS. 

Inoculate  a  male  guinea-pig  in  the  peritoneal  cavity  with  a 
solution  of  the  suspected  material  or  culture,  making  the  inoculation 
direct  in  the  middle  line  of  the  abdomen,  otherwise  other  bacteria 
may  be  introduced  into  the  vesiculae  seminalis,  and  cause  orchitis,  etc., 
or  introduce  a  piece  of  the  suspected  tissue.  If  the  material 
inoculated  is  from  a  genuine  case  of  glanders,  the  testicles  commence 
to  swell  in  thirty  hours,  and  the  skin  over  them  becomes  hypersemic, 
shiny,  and  finally  desquamates,  evidence  of  the  formation  of  pus 
appears,  and  the  purulent  orchitis  often  breaks  through  the  skin. 
The  diagnostic  symptom  is  the  tumefaction  of  the  testicles. 

Mallein. — This  consists  of  the  filtered  products  of  the  glanders 
bacillus, — a  group  of  compounds  bearing  a  similar  relation  to  glanders 
that  tuberculin  bears  to  tuberculosis.  It  is  prepared  from  old  glycerine 
bouillon  cultures  of  the  glanders  bacillus  by  steaming  them  for  several 
hours  in  the  sterilizer,  or  in  the  autoclave  for  fifteen  minutes  at  115°  C., 
and  filtering  through  unglazed  porcelain,  the  filtrate  being  concentrated 
one-sixth  its  volume,  and  mixed  with  an  equal  volume  of  J  per  cent, 
solution  of  carbolic  acid.  This  yields  an  active  mallein,  the  dose  being 
1  c.c.,  and  it  gives  good  reactions. 


134  SPECIAL  BACTERIOLOGY 

The  Diagnosis  of  Glanders  with  Mullein,  according  to  Hunting,  vide 
Veterinary  Record)  December  4th,  1897. — '  With  mallein  diagnosis  is  easy, 
and  in  ninety-nine  out  of  a  hundred  cases  is  certain.  An  injection  of  mallein 
under  the  skin  of  a  healthy  horse  has  no  effect,  or  at  most  it  produces  a  swelling 
as  big  as  a  watch  at  the  point  of  injection.  An  injection  into  a  glandered 
horse  produces  two  reactions, — a  large  and  painful  swelling  at  the  point  oj 
injection,  and  a  rise  of  temperature  to  104°  or  even  106°  Fahr.  The  indica- 
tions of  mallein  are  not  always  so  exact  that  it  can  be  used  without  brains.  It 
will  not  do  to  say  no  horse  is  glandered  until  the  temperature  rises  4°  and  a 
swelling  appears  within  twenty-four  hours  measuring  Jive  inches  across.  Some- 
times the  local  swelling  is  less,  and  sometimes  the  temperature  does  not  rise 
much.  When  the  temperature  is  already  103°  F.  a  rise  may  not  take  place  at 
all ;  but  in  such  a  case  a  painful  swelling  at  the  point  of  injection  is  conclusive 
evidence.  In  hundreds  of  cases  I  have  proved  the  trustworthiness  of  mallein, 
when  no  outward  sign  of  disease  existed,  by  post-mortem  residts.  In  hundreds 
of  healthy  horses  1  have  known  it  used  without  one  ill  effect.' 

The  above  conclusions  are  of  immense  practical  and  diagnos  tic  value 
owing  to  Hunting's  extensive  practical  experience  with  glanders  and 
the  use  of  mallein  in  London. 

M.  Nocard  recommends  that  only  animals  presenting  clinical  signs 
reacting  to  the  mallein  test  should  be  slaughtered.  The  other  animals 
should  be  isolated  and  submitted  every  month  or  two  months  to  the 
mallein  test,  and  when  they  have  supported  two  tests  without  reacting, 
they  can  be  placed  at  the  free  disposal  of  the  owners,  for  they  will  then 
have  completely  and  definitely  recovered  from  the  glanders  lesions 
which  they  carried  in  their  lungs.  M.  Nocard  further  states  that 
recovery  is  an  occurrence  far  from  being  rare.  The  glanders  nodules 
found  on  post-mortem  examination  of  such  cases  did  not  produce 
disease  when  inoculated  into  susceptible  animals,  nor  could  any  diag- 
nostic cultures  be  obtained. 


BACILLUS  ORCHITICUS. 

Found  by  Kutscher  in  the  nasal  discharge  of  a  horse  affected  with 
glanders. 

Microscopical  Appearances. — Similar  to  the  glanders  bacillus. 

Motility. — Non-motile. 

Staining  Reactions.  —  With  ordinary  stains  and  by  the  Gram 
method. 

Vitality. — Killed  by  exposure  to  55°  C.  for  five  minutes. 

Biological  Characters. — It  grows  on  all  the  ordinary  media  except 
milk. 


BACILLUS  ORCHITICUS  135 

On  Gelatine  Plates  it  forms  colonies  that  resemble  old  colonies  of  the 
bacillus  of  Asiatic  cholera  ;  liquefaction  occurs  somewhat  rapidly  at  22°  C. 

On  A  gar,  thick  white  tufts. 

On  Blood  Serum  an  orange-yellow  pigment  is  often  formed;  the 
medium  is  liquefied. 

In  Bouillon  and  Peptone  Solution  small  flakes  are  formed,  the  medium 
very  seldom  becoming  clouded. 

Pathogenesis.  —  Guinea-pigs  (male)  when  inoculated  intraperi- 
toneally  with  a  small  quantity  of  the  virus,  exhibit  swelling  of  the 
testicles  in  forty-eight  hours,  and  generally  die  in  four  to  five  days. 
The  principal  lesions  are  nodules  in  the  mesentery  and  testicles  (rarely 
in  the  abdominal  organs).  Large  doses  caused  death  in  one  to  three 
days  with  more  pronounced  changes  in  the  peritoneal  cavity.  Small 
doses  introduced  subcutaneously  caused  death  in  one  to  two  days,  with 
an  extensive  oedema  affecting  the  whole  abdominal  wall.  Guinea-pigs 
that  recovered  were  found  later  to  be  immune  to  further  infection. 
Mice  inoculated  subcutaneously  with  small  doses  die  in  four  to  seven 
days,  an  abscess  developing,  the  surrounding  tissue  being  cedematous, 
and  infiltrated  with  small  haemorrhages.  The  bacilli  were  only  present 
in  the  pus  from  the  abscess,  and  frequently  within  the  leucocytes. 
Intraperitoneal  inoculation  produced  death  in  the  same  time,  numerous 
nodules  being  formed  on  the  peritoneum  ;  the  liver  and  spleen  being 
seldom  affected.  Intrapulmonary  injection  caused  the  formation  of  a 
watery  hsemorrhagic  effusion  into  the  serous  cavities  of  the  thorax,  and 
the  development  of  numerous  grey  nodules  on  the  pleurae,  associated 
with  small  lobular  pneumonic  centres. 

Rabbits  are  not  so  susceptible  to  infection,  while  chickens  and  pigeons 
are  immune. 

Differential  Diagnosis. — With  the  Gram  method  of  staining  the 
bacillus  of  glanders  gives  negative  results,  while  the  Bacillus  orchiticus 
yields  positive  results. 

EPIZOOTIC    LYMPHANGITIS,    OR    AFRICAN    FARCY. 

An  organism  discribed  as  the  '  Cryptococcus  of  Rivolta,'  a  species  of 
micrococcus  measuring  3  to  4  /A  in  diameter,  slightly  ovoid  and  somewhat 
pointed  at  one  of  its  extremities,  has  been  found  in  the  pus  and  lesions 
of  this  disease  by  Rivolta  and  Nocard.  It  stains  by  the  Gram,  Weigert, 
and  Kiihne  methods. 

The  dimensions  and  refringence  exhibited  by  the  organism  are  such 
that  it  is  impossible,  even  in  unstained  specimens,  to  mistake  it  for  any 
other  element  (Nocard). 

Ulcers  resembling  those  of  acute  glanders  have  been  found  on  the 
nasal  mucosa  of  animals  affected  with  epizootic  lymphangitis.  Nocard, 
however,  found  the  above  cryptococcus  in  these  lesions,  and  thus  affirmed 


136  SPECIAL  BACTERIOLOGY 

their  relation    to    lymphangitis    and    not    to    glanders,    the    bacillus   of 
glanders  being,  moreover,  absent. 


TUBERCULOSIS. 

The  infectious  nature  of  this  disease  was  first  demonstrated  by 
Villemin  -in  1865,  when  he  communicated  the  disease  to  healthy 
experiment  animals  with  tuberculous  material.  Cohnheim  confirmed 
these  experiments  by  inoculations  into  the  anterior  chamber  of  the 
eyes  of  experiment  animals.  In  1882  Koch  discovered  the  Bacillus 
tuberculosis,  which  is  now  known  as  the  cause  of  mammalian  tuber- 
culosis. 

Microscopical  Appearances. — Koch's  bacilli  are  small,  thin  rods, 
varying  in  size  from  0'2  to  0*4  /x,  broad,  to  3  to  4  p  long;  they  are 
slightly  bent,  generally  occur  singly,  but  in  cultures  sometimes  form 
chains  of  four  to  six  individuals  ;  in  rare  cases  they  exhibit  a  club-like 
swelling  at  one  end  and  branches,  whereby  a  certain  relationship  with 
the  actinomyces  group  has  been  ascribed  to  them. 

Motility. — Non-motile. 

Spore  Formation. — The  clear  spaces  present  in  stained  specimens 
have  been  described  by  some  authorities  as  spores,  and  by  others  as  due 
to  degenerative  processes. 

Staining  Reactions. — The  bacilli  stain  with  difficulty,  but  once  they 
are  stained  they  retain  the  dye  with  great  tenacity.  The  results  with 
the  Gram  and  Cladius  staining  methods  are  positive.  For  the  special 
methods  of  preparing  and  staining  cover-glass  specimens  and  sections 
see  Technique,  §§  13,  14,  40. 

According  to  Koch's  recent  investigations,  tubercle  bacilli  contain 
two  solid  fatty  acids,  one  of  which  is  soluble  in  reduced  alcohol  and 
saponified  by  caustic  soda ;  the  other  is  not  saponified,  and  is  only  soluble 
in  boiling  absolute  alcohol  and  ether.  Both  of  these  fatty  acids  are 
stained  by  the  specific  tubercle  bacilli  stains ;  but  by  the  process  of 
decolorization,  the  one  soluble  in  alcohol  gives  up  the  stain,  while  the 
other  retains  it ;  thus  the  acid  fixes  the  staining  material  and  accounts  for 
the  staining  reaction  (see  Photomicrograph  of  tubercle  bacilli  in  sputum, 
Fig.  47).  It  is  possible  with  a  warm  solution  of  caustic  soda  to  remove 
the  fatty  acids  out  of  the  body  of  the  bacilli,  and  observe  by  a  micro- 
scopical examination  how  they  pass  out  in  the  form  of  colourless  drops, 
and  coalesce  into  larger  drops.  According  to  Koch,  these  fatty  acids 
form  a  continuous  layer  in  the  bodies  of  the  bacilli,  thus  providing  them 
with  a  protection  against  external  influences. 

This  peculiar  micro-chemical  staining  reaction  found  in  the  case  of 


TUBERCULOSIS  137 

the  Bacillus  tuberculosis  is  not  confined  to  that  organism  alone,  as  other 
species  of  bacilli,  when  similarly  treated,  react  in  the  same  way,  e.g. :  — 

1.  The  smegma  bacillus,  located  in  the  smegma,  often  seen  beneath 
the  prepuce  and  upon  the  vulva,  both  normally  and  in  disease. 

2.  Lustgartens  bacillus  of  syphilis,  found   principally  in  the  primary 
sores  associated  with  that  disease. 

3.  The  bacillus  of  leprosy. 

1  4.   The  acid-resisting  bacteria  found  in  butter. 

Hueppe  differentiates  the  first  three  organisms  and  the  Bacillus 
tuberculosis  as  follows  : — 

1.  Treat  the  preparation,  stained  with  carbol  fuchsin,  with  sulphuric 
acid,  and  the  syphilis  bacillus  is  decolorized  almost  instantaneously. 

2.  If  not  at  once  decolorized,  treat  with  alcohol,  and  if  it  is  the 
smegma  bacillus,  it  will  lose  its  colour. 

3.  If  it  is  still  not  decolorized,  it  is  either  the  leprosy  or  tubercle 
bacillus.     According  to  Baumgarten,  the  Lepra  bacillus  is  stained  by  an 
exposure  of  six  or  seven  minutes  to  a  cold,  saturated,  watery  solution  of 
fuchsin,  and  retains  the  stain  when  subsequently  treated  with  acid  alcohol 
(nitric  acid  1   part,  alcohol  10).     When  treated  for  the  same  length  of 
time,  the  bacillus  of  tuberculosis  does  not  ordinarily  become  stained. 

Biological  Characters. — It  is  very  difficult  to  obtain  a  pure  culture 
of  tubercle  bacilli,  because  they  grow  extraordinarily  slowly,  and  require 
for  their  development  an  incubator  temperature  of — minimum,  29° ; 
maximum,  41° ;  and  optimum,  37  to  38°  C. 

The  Koch  bacillus  grows  well  on  blood  serum,  4  to  6  per  cent,  glycerine 
agar,  and  in  glycerine  bouillon.  The  isolation  of  the  tubercle  bacillus 
from  the  mixture  of  bacteria  in  tubercular  sputum  by  means  of  glycerine 
agar  plates  is  almost  impossible ;  on  account  of  the  tubercle  bacilli 
growing  so  slowly,  the  other  bacterial  colonies  outgrow  and  overwhelm 
them  easily.  It  is  only  possible  to  obtain  a  pure  culture  when  the 
material  is  quite  pure  at  the  commencement.  The  following  is  the 
method  of  procedure  : — 

Inoculate  some  guinea-pigs  (which  are  very  susceptible)  with  material 
containing  tubercle  bacilli.  In  about  four  weeks  the  first  inoculated 
animal  dies,  the  autopsy  revealing  a  well-marked  tuberculosis  of  the 
internal  organs.  One  of  the  other  guinea-pigs  is  now  killed,  the  skin  dis- 
infected with  warm  water  and  sublimate  solution  (1  to  1000),  and  removed 
with  a  knife  heated  in  the  Bunsen  flame.  The  peritoneum  is  opened 
with  other  sterilized  instruments,  and  the  spleen  removed  with  forceps 
previously  heated  in  the  flame.  By  this  mode  of  infection  the  spleen  is 
usually  the  most  strongly  affected.  A  portion  containing  the  tubercles 
is  removed  from  the  spleen  with  sterilized  forceps,  and  pressed  between 
two  aseptic  scalpels  or  glass  slides  in  order  to  obtain  material  containing 
bacilli,  which  is  conveyed  by  means  of  a  stout  sterile  platinum  needle 


1S8  SPECIAL  BACTERIOLOGY 

on  to  the  surface  of  bjood  serum  medium.  The  whole  process  must  be 
accomplished  quickly  and  with  the  greatest  cleanliness,  because  if 
another  germ  enters  the  serum  tube,  it  will  soon  outgrow  the  tubercle 
bacillus.  As  a  greater  precaution,  several  cultivations  ought  to  be 
instituted  at  the  same  time.  The  cultures  remaining  a  long  time 
in  the  incubator  at  37°  to  38°  C.,  they  must  be  closed  with  indiarubber 
caps  previously  sterilized  in  sublimate  solution,  or  the  superfluous  over- 
hanging cotton -plug  burnt  off  in  the  flame,  and  the  tube  closed  with 
melted  paraffin  or  sealing-wax.  Without  these  precautions  the  water  of 
condensation  will  be  all  absorbed  arid  the  culture  medium  dried  up. 

When  the  inoculation  is  successful,  signs  of  growth  are  observed  in 
fourteen  days  in  the  blood  serum  tubes.  Small,  grey,  dry  scales  develop, 
which  when  examined  under  a  low  power  appear  composed  of  delicate 
twisted  lines.  The  development  goes  on  slowly,  and  in  four  to  six 
weeks  other  tubes  can  be  inoculated  from  the  original  culture.  The 
growth  of  the  second  generation  requires  two  weeks  before  it  is  distinct. 
Later  generations,  usually  the  fifth  or  sixth,  grow  more  luxuriantly  and 
quicker,  especially  when  a  special  incubator  is  used,  provided  with  an 
atmosphere  of  steam,  so  that  the  use  of  indiarubber  caps,  etc.,  can  be 
dispensed  with.  Under  such  conditions,  in  seven  to  fourteen  days  the 
whole  of  the  surface  of  the  serum  medium  is  covered  with  the  character- 
istic dry  scales.  From  the  fifth  serum  generation  it  is  easy  to  obtain 
cultures  on  glycerine  agar.  On  this  medium  the  development  is  much 
more  abundant,  the  bacilli  forming  a  greyish  dry  coating  of  brittle, 
curly,  slightly  elevated  fragments  (see  Photograph,  Fig.  48). 

This  coating  growing  downwards,  covers  any  water  of  condensa- 
tion present  without  clouding  it,  and  when  the  culture  is  left  long 
enough  in  the  incubator  it  grows  a  considerable  distance  up  the  free 
sides  of  the  test-tubes,  where  no  nutrient  medium  is  present.  Veal 
bouillon,  with  6  per  cent,  glycerine  added,  and  filled  in  Erlenmeyer 
flasks,  is  the  best  fluid  medium.  When  inoculating  this  bouillon  it  is 
necessary  to  place  the  dry  scales  in  the  fluid  so  that  they  swim  on  the 
surface,  as  the  tubercle  bacilli,  being  susceptible  to  oxygen,  develop  only 
in  the  upper  portions  of  the  medium  where  the  air  has  sufficient  access. 
On  the  surface  of  veal  6  per  cent,  glycerine  bouillon  the  tubercle  bacilli 
form  a  membranous  surface  growth  which  exhibits  the  same  char- 
acteristics as  the  coating  on  glycerine  agar.  Under  favourable 
circumstances,  in  a  few  weeks,  sometimes  in  ten  days,  the  growth  extends 
a  considerable  distance  up  the  walls  of  the  tube ;  the  lower  portion  of 
the  bouillon  remains  perfectly  clear,  which  is  a  characteristic  of  the  growth 
of  the  tubercle  bacillus. 

On  Potatoes)  the  under  ends  of  which  project  into  a  solution  contain- 
ing 5  per  cent,  of  glycerine  and  5  per  cent,  of  NaCl.,  placed  in  the 
bottom  of  the  tube,  the  tubercle  bacilli  develop  very  well,  forming  on 
the  surface  of  the  potatoes  thick  warty  tufts,  the  glycerine  solution 


Fi«i.  47. — B.  Tuberculosis.    Cover-glass  specimen  prepared  from 
sputum.     Ehrlich's  method.     X 1000. 


FIG.  48.— B.  Tuberculosis. 
Glycerine  agar  culture. 


Ft<;.  4'.'.— B.  Tuberculosis.    Cover-glass  specimen  from  purr 
culture  on  glycerine  agar.     Ehrlich's  method,     x  1000. 


IT.  llowkill,  t\R.U.V.S.,  1'hutn.,  Edhil»trr/1i,  1SU8. 


TUBERCULOSIS  139 

remaining  clear.  The  potato  culture  medium  is  prepared  according  to 
Roux  and  Globig's  method  (see  Technique,  p.  49).  In  1882  Koch  and 
Kitasato  devised  a  means  of  obtaining  cultures  of  tubercle  bacilli  direct 
from  the  sputum  of  phthisical  patients.  They  washed  out  the  patient's 
mouth  with  an  antiseptic  gargle,  and  emptied  the  expectorate  into  a 
Petri-dish.  One  of  the  expectorated  masses  of  sputum  is  washed  in 
several  changes  of  sterile  water  to  free  the  exterior  of  bacteria ;  out  of 
the  middle  of  the  washed  sputum  a  portion  is  removed  with  the  platinum 
needle,  and  stroke  cultures  prepared  on  the  surface  of  blood  serum. 
The  developing  cultures  exhibit  a  somewhat  different  appearance  to 
those  cultivated  from  the  bodies  of  animals;  they  appear  as  round, 
whitish,  transparent  colonies,  but  the  later  generations  grow  in  the  same 
manner  as  those  previously  described.  The  bacilli  can  be  cultivated 
through  many  generations  for  several  years  without  injuring  their  power 
of  growth,  although  the  older  cultures  become  less  virulent.  (For  Photo- 
micrograph of  specimen  from  pure  culture,  see  Fig.  49.) 

Vitality. — The  Bacillus  tuberculosis  is  destroyed  by  heating  for  ten 
minutes  at  70°  C.,  one  minute  at  95°  C.,  one  hour  at  60°  C.,  and  four 
hours  at  55°  C.  They  only  resist  the  action  of  direct  sunlight  for  from 
some  minutes  to  some  hours,  according  to  the  thickness  of  the  growth. 
Exposed  to  diffuse  daylight  they  are  killed  in  a  week. 

Pathogenesis. — None  of  the  domestic  animals  are  immune.  The 
guinea-pig  is  the  most  susceptible  of  all  the  experimental  animals,  a 
very  small  quantity  of  tubercle  bacilli  being  sufficient  for  their 
infection  ;  next  come  the  rabbit  and  the  field  mouse ;  less  susceptible, 
and  still  far  from  immune,  are  white  mice  and  dogs.  Young  animals 
exhibit  a  greater  predisposition  for  tuberculosis  than  older  animals. 
Typical  tubercular  lesions  are  produced  in  guinea-pigs,  rabbits,  and 
field  mice  by  subcutaneous  injection,  inoculation  into  the  anterior 
chamber  of  the  eye,  intraperitoneal  and  intravenous  injection,  or  by 
inhalation  of  moist  powdered  tubercle  bacilli. 

Susceptible  animals  infected  by  feeding  with  tubercular  matter  die 
of  abdominal  tuberculosis. 

Baumgarten  inoculated  animals  in  the  anterior  chamber  of  the 
eye,  and  in  three  days  found  the  bacilli  in  the  auricular  lymph-glands. 
Intravenous  injection  produces  a  generalized  miliary  tuberculosis. 
The  experiments  of  Schiiller  are  of  especial  interest  in  relation  to 
many  localized  tubercular  diseases  of  man ;  he  injected  tubercular 
material  in  a  suitable  part  of  an  animal,  then  produced  an  injury  in 
the  region  of  the  knee-joint,  and  observed  that  the  infection  became 
localized  at  that  point.  Dead  tubercle  bacilli  produce  pyogenic 
results  ;  they  are  positive  chemotactic,  drawing  the  leucocytes  towards 


140  SPECIAL  BACTERIOLOGY 

them.  When  dead  bacilli  are  injected  intravenously  into  rabbits,  in 
the  animals  killed,  after  some  time,  small  tubercles  are  found  through- 
out the  lungs  and  liver,  formed  of  round  cells,  epithelioid  cells,  giant 
cells,  and  dead  tubercle  bacilli,  which  cannot  be  distinguished  from 
the  genuine  Bacillus  tuberculosis.  Baumgarten  considers  that  the 
dead  tubercle  bacilli  produce  a  tuberculosis  similar  to  that  produced 
by  a  foreign  body  (pseudo-tuberculosis). 

Instances  in  which  physicians  and  veterinary  surgeons  have  con- 
tracted the  disease,  in  making  autopsies  of  diseased  men  or  animals, 
are  incontestable,  although  fortunately  rare.  The  virus  in  such  cases 
gains  entrance  by  some  cutaneous  wound,  causing  at  first  a  more  or 
less  limited  cutaneous  tuberculosis,  which  later  may  become  generalized. 


BOVINE  TUBERCULOSIS. 

According  to  most  authorities,  tuberculosis  of  the  lungs  (see 
Photomicrograph,  Fig.  50)  is  the  most  frequent  of  all  the  primary 
forms  of  this  disease  in  cattle,  infection  being  caused  by  dried  tuber- 
cular matter  inhaled  into  the  lungs.  Primary  tuberculosis  also 
occurs  in  the  lymph-glands  of  the  head  and  neck,  in  the  mesenteric 
glands,  the  intestines,  the  liver,  the  genital  organs,  and  the  udder. 
Bang  is  inclined  to  believe  that  the  udder  is  now  and  then  primarily 
affected  in  animals  that  are  in  very  good  condition.  Eber  reports  a 
case  of  primary  tuberculosis  of  the  penis,  and  cases  involving  the 
vagina  and  vulva.  Finally,  generalized  infection,  due  to  the  dissemi- 
nation of  tubercle  bacilli  through  the  blood,  occurs  in  two  forms : 
(1 )  The  acute  form,  when  large  numbers  of  bacilli  have  escaped  into 
the  circulation,  whereby  numerous  tubercular  foci  appear  in  various 
organs ;  (2)  The  chronic  form,  as  seen  in  old  cows  affected  with 
tuberculosis  for  many  years.  A  few  cases  of  congenital  tuberculosis 
have  been  recorded  in  calves.  Johne  found  tubercle  bacilli  twice  in 
the  organs  of  embryos.  The  writer  found  a  case  of  tubercular 
meningitis  in  a  calf  in  California.  The  mother  of  this  calf  was 
tested  with  tuberculin  and  reacted,  the  post-mortem  revealing 
tubercular  lesions,  one  ovary  being  affected. 

King  mentions  a  case  of  a  cow  giving  birth  to  twin  calves.  The 
viscera  of  both,  when  submitted  to  examination,  proved  tubercular. 
The  cow  was  not  tested  with  tuberculin,  being  considered,  from 
apparent  symptoms,  affected  with  generalized  disease. — (Proceeding's 
of  National  Veterinary  Association^  Leeds,  1898.) 

The  bacillus  of  Koch,  according  to  Nocard,  is   only  in   very  ex- 


BOVINE  TUBERCULOSIS  141 

ceptional  cases  transmitted  from  mother  to  foetus ;  the  predisposition 
to  receive  and  develop  the  germ  is,  however,  hereditary.  Infection 
rarely  takes  place  while  diseased  and  healthy  cattle  are  pastured 
together  in  the  open.  Nocard  and  other  observers  frequently  point 
to  the  fact  of  the  animals  being  infected  while  standing  in  stalls 
adjacent  to  the  coughing  '  piner,'  the  disease  often  extending  along 
one  side  of  the  byre,  while  the  cows  on  the  other  side  may  remain 
sound,  and  it  is  also  observed  that  the  animals  longest  stabled  furnish 
the  largest  proportion  of  cases.  While  the  author  was  Inspector  for 
the  city  and  county  of  San  Francisco,  the  tubercular  seizures,  with  a 
few  exceptions,  were  all  dairy  cows,  tubercular  lesions  in  range  cattle 
being  hardly  ever  observed. 

Nocard  is  against  the  total  seizure  of  the  carcases  of  cattle  which 
have  localized  tuberculosis,  but  are  otherwise  in  good  condition.  He 
insists  that  the  bacilli  of  Koch,  the  true  infectious  material,  does  not 
exist  either  in  the  blood  or  in  the  muscles,  excepting  for  very  brief 
periods,  and  in  cases  of  advanced  general  tuberculosis.  In  some  of 
Nocard's  experiments  with  meat  from  cases  of  generalized  tuberculosis, 
he  found  that  although  it  had  no  bad  effects  when  eaten,  the  ex- 
pressed juice  produced  tuberculosis  in  one  or  two  of  the  guinea-pigs 
when  injected  into  the  peritoneum.  It  is  generally  admitted  that 
the  milk  of  tuberculous  cows  is  greatly  more  liable  than  their  flesh  to 
produce  human  tuberculosis.  That  this  is  so  is  very  conclusively 
proved  by  the  fact  that  abdominal  tubercle  is  practically  confined  to 
children  under  five  years  whose  food  consists  very  largely  of  raw  milk, 
while  the  incidence  is  greatest  in  cases  of  infants  hand-fed.  Nothing 
except  milk  infection  can  account  for  the  terrible  prevalence  among 
children  of  tabes  mesenterica  and  allied  tubercular  disease.  The 
milk  is  not,  however,  always  virulent.  It  is  only  so  when  the  udder 
is  infiltrated  with  tubercular  nodules.  Nocard  states  that  in  his 
experience  he  never  found  the  milk  virulent  when  the  udder  was  free 
from  tuberculous  lesions,  and  out  of  fifty -four  cows  seized  for  general 
tuberculosis  which  he  specially  examined,  only  three  had  tubercular 
lesions  in  the  udder.  At  Copenhagen  the  proportion  is  still  lower. 
Bang  estimates  it  at  less  than  3  per  cent,  of  the  number  of  tuber- 
culous cows.  However,  the  difficulty  of  deciding  as  to  the  non- 
existence  of  tubercle  in  the  mammary  gland  justifies  the  milk  from 
suspicious  cases  being  excluded  from  consumption.  (For  the  special 
methods  of  examining  and  staining  tubercle  bacilli  in  milk,  see 
Technique,  §  20.) 

The  question  of  living  tubercle  bacilli  existing  in  ordinary  market 
butter  has  led  to  considerable  investigation  since  Obermuller  stated 


142  SPECIAL  BACTERIOLOGY    . 

that  in  fourteen  samples  of  butter  he  found  genuine  tubercle  bacilli, 
capable  of  causing  infection.  Rabinowitsch,  in  eighty  samples  of 
butter  that  he  examined,  did  not  find  the  Koch  bacilli  once,  and  he 
considered  that  the  positive  results  obtained  by  others  were  due  to 
the  acid-resisting  tubercle-like  bacilli  previously  discovered  by  him 
being  mistaken  for  genuine  tubercle  bacilli. 

Petri's  results  occupy  an  intermediate  position  between  those  of 
Obermiiller  and  Rabinowitsch.  He  found  genuine  tubercle  bacilli 
in  30  per  cent.,  and  the  acid-resisting  tubercle-like  bacilli  in  60  per 
cent,  of  the  samples  examined. 

Horman  and  Morgenroth  conducted  a  series  of  investigations 
recently,  and  in  ten  samples  of  market  butter  examined  they  found 
genuine  tubercle  bacilli  in  three  of  the  samples  (see  Photomicrograph 
of  same,  Plate  III.,  Fig.  18),  and  in  some  of  the  samples,  Rabino- 
witsch's  acid-resisting  bacilli ;  and  in  one  of  the  experiments  the 
resisting  bacteria  and  genuine  tubercle  bacilli  were  found  associated 
together.  Glycerine  agar  was  of  no  use  as  a  culture  media  ;  blood 
serum,  with  the  addition  of  5  per  cent,  of  glycerine,  being  found  the 
best  medium  in  these  investigations. 

The  acid-resisting  tubercle-like  bacilli  found  in  butter  are 
described  by  Horman  and  Morgenroth  as  follows : — 

Microscopical  Appearances. — Slender  rods,  similar  to  the  Koch's 
bacillus  of  tuberculosis  in  form,  slightly  bent,  and  sometimes  the  end  of 
the  rod  is  thickened. 

Motility. — Non-motile. 

Staining  Reactions. — When  stained  by  Gunther's  method  for 
tubercle  bacilli,  they  are  not  decolorized,  but  their  resistance  is  not  so 
pronounced  as  with  Koch's  bacilli,  as  on  the  edges  of  the  cover-glass 
and  contact  specimen  of  colonies  many  of  the  rods  are  stained  slightly 
blue.  In  old  cultures  unstained  portions  in  the  stained  rods  were  often 
observed.  The  reaction  with  the  Gram  method  is  positive. 

Biological  Characteristics.  —  On  Gelatine  Plates  the  growth 
is  very  slow.  The  deep-lying  colonies  are  round,  glistening,  of 
a  light-yellow  colour,  and  finely  granular  throughout.  The 
superficial  colonies  are  also  round,  transparent,  and  possess  irregular, 
finely  serrated  margins. 

The  gelatine  is  not  liquefied. 

In  Gelatine  Stab  Cultures  a  growth  occurs  along  the  middle  track,  and 
after  a  long  time  a  thick  coating  develops  on  the  surface. 

On  Agar  oblique  surface  Cultures  in  twenty-four  hours,  at  incubator 
temperature,  a  white,  creamy  coating  develops  on  the  surface  of  the 


FIG.  50.— B.  Tuberculosis.    Cover-glass  specimen,  lung  of  a  cow. 
Ehrlich's  method.     X  1000. 


FIG.  51.— B.  Tuberculosis.     Section  of  mesenteric  gland 
of  the  horse.    Ehrlich's  method.     X  600. 


[T.  Lowhill,  F.R.C.V.S.,  Photo.,  Edinburgh,  1808. 


BOVINE  TUBERCULOSIS  143 

water  of  condensation,  a  thin  membrane  which  passes  on  to  the  walls  ot 
the  tube.  In  old  cultures  the  coating  is  frequently  very  much  corru- 
gated, and  sometimes  also  exhibits  a  yellow,  orange,  or  light-brown 
colour. 

Bouillon  or  Glycerine  Bouillon  becomes  clouded  with  a  sediment,  and 
in  forty-eight  hours  a  membrane  forms  on  the  surface,  which  sinks  very 
easily  to  the  bottom  of  the  medium,  if  shaken,  after  which  another 
membrane  forms,  which  passes  up  the  sides  of  the  tube.  A  slight 
formation  of  indol  was  observed  in  bouillon  cultures. 

On  Potatoes  the  growth  is  rapid  and  plentiful,  exhibiting  a  thick, 
greyish,  moist  coating  in  twenty-four  hours. 

Sterile  Milk  is  not  altered  by  the  growth  of  the  bacteria,  but  a 
yellowish-white  membrane  forms  on  the  surface. 

The  morpho-biological  characteristics  above  mentioned  correspond 
to  those  of  the  acid-resisting  bacteria  discovered  by  Rabinowitsch. 

Pathogenesis. — These  acid-resisting  bacteria  produced  disease 
processes  in  guinea-pigs  when  injected  into  the  peritoneum. 

The  liver  was  the  only  organ  in  which  the  lesions  bore  a  distinct 
resemblance  to  those  of  tuberculosis.  The  tubercles  were  of  a  greyish- 
white  colour,  and  penetrated  from  the  surface  of  the  liver  into  the 
parenchyma,  easily  detached  from  the  surrounding  tissue.  Small  to 
pinhead-sized  yellow  spotted  foci  were  also  observed  on  the  surface 
as  well  as  in  sections  of  the  organs.  The  spleen,  on  the  contrary, 
never  exhibited  the  appearance  of  a  genuine  tuberculous  spleen.  The 
enlargement,  dark  colour,  and  characteristic  marbling  were  awanting. 
In  the  most  of  cases  extensive  peritoneal  adhesions  were  present. 
The  mesenteric  lymph-glands  were  not  very  much  enlarged.  In  one 
case  softening  was  observed ;  it  was  of  a  purulent  nature,  not  caseous. 
These  bacteria  frequently  cause  peritoneal  lesions  which  exhibit  a 
decided  tendency  to  recovery.  In  cases  where  reparative  changes 
were  observed,  the  acid-resisting  bacteria  appeared  to  die  and  dis- 
appear, because  in  cover-glass  specimens  and  sections  from  some  of 
the  guinea-pigs  experimented  with,  the  bacteria  could  no  longer  be 
detected.  Two  chickens  and  one  dog  injected  intraperitoneally  with 
a  pure  culture  remained  unaffected.  According  to  Rabinowitsch, 
rabbits  are  refractory.  Injection  into  the  anterior  chamber  of  the 
rabbit's  eye  caused  inflammatory  changes  which  did  not  persist  very 
long. 

HORMAN   AND    MORGENRATITS   METHOD   OF   DEMON- 
STRATING TUBERCLE  BACILLI  IN  BUTTER. 

1.  Inject  4  to  5  c.c.  of  butter  melted  at  37°  C.,  and  thoroughly  mixed 
into  the  peritoneal  cavity  of  three  guinea-pigs.  (Allowing  the  butter  to 


144  SPECIAL  BACTERIOLOGY 

stand  from  twelve  to  twenty-four  hours  at  34°  C.,  as  recommended  by 
Rabinowitsch,  is  unnecessary.) 

2.  From  the  organic  lesions  of  the  guinea-pig  that  die  or  are  killed 
in  four  to  six  weeks  cultures  are  instituted  on,  at  the  least,  eight  to  ten 
tubes  of  blood  serum,  and  at  the  same  time  pieces  of  the  organs  are 
inserted  into  the  peritoneal  cavities  of  two  guinea-pigs  and  one  rabbit. 

3.  These  latter  animals  are  killed  not  later  than  four  weeks,  and  from 
any  existing  lesions  blood  serum  cultures  instituted. 

Conclusions. — (1.)  It  has  been  observed  that  genuine  Koch  tubercle 
bacilli  are  often  present  in  butter. 

(2.)  Acid-resisting  forms  of  bacteria  are  also  found  in  butter,  which 
cause  disease  processes  in  guinea-pigs.  These  changes  were,  however 
in  the  above  authors'  experiments  not  such  that  they  could  not  be  dis- 
tinguished from  the  changes  produced  by  genuine  tubercle  bacilli. 


EQUINE   TUBERCULOSIS. 

In  the  horse  this  disease  manifests  itself  in  two  forms. 

1.  The  Abdominal  Form,  which  seems  to  be  the  more    frequent,  is 
characterised    by    confluent    lesions    in    the    spleen,    sub-lumbar    and 
mesenteric  glands  (for  section  of  same    showing   tubercle   bacilli,  see 
Photomicrograph,    Fig.    51),   liver,    and   intestinal    mucous    membrane. 
According   to    Nocard,  infection    seems   to  take  place    by  way  of  the 
alimentary  canal.        When  the   lungs  are  invaded  as  a  sequel  of  the 
abdominal  form,  the  lesions    appear   of  recent   origin,  and    consist   of 
a    diffuse    infiltration    of    the    interlobular    connective    tissue    without 
apparent  tubercles,  caverns,  or  centres  of  softening,  which,  according  to 
the  above  authority,  accounts  for  the  absence  of  cough  and  discharge  or 
expectoration,  and  the  non-transmission  of  the  disease  to  other  horses 
in  the  same  stable. 

Nocard  also  states  that  in  advanced  cases  an  abundant  polyuria, 
lasting  for  several  weeks,  occurs.  Koch's  bacilli  are  extremely  abundant 
in  the  lesions,  and  of  very  great  length.  Nocard  considers  that  the 
bacillus  of  equine  abdominal  tuberculosis  is  more  closely  allied  to  the 
bacillus  of  avian  tuberculosis  than  to  the  bacillus  of  mammalian  tuber- 
culosis. 

2.  The  Thoracic  Form. — In  this  type  the  disease  seems  to  originate 
primarily  in  the  lungs,  because  these  organs  and  the  bronchial  glands 
are  most  severely  affected.     A  genuine  acute  miliary  tuberculosis  some- 
times occurs ;  at  other  times  the  parenchyma  of  the  lungs  is  studded 
with  small  abscesses  with  a  fibrous  capsule,  enclosing  pus  that  is  very 
rich  in  bacilli.     Nocard  claims  that  the  distinction  drawn  between  the 
two  types  of  equine  tuberculosis,  on  clinical  and  pathological  grounds,  is 
confirmed   by  the   determination   of   the    causal    agent,  for   while   the 


TUBERCULOSIS  OF  SWINE  145 

thoracic  form  is  referable  to  human  tuberculosis,  the  abdominal  form, 
as  already  mentioned,  is  more  closely  allied  to  the  bacillus  of  avian 
tuberculosis. 

CANINE  TUBERCULOSIS. 

In  the  dog  the  disease  also  occurs  in  two  forms,  the  abdominal  and 
the  thoracic.  Many  cases  have  been  recorded,  the  principal  lesions 
mentioned  occurring  in  the  liver,  hepatic  and  mesenteric  glands,  as  well 
as  generalized  tuberculosis  of  both  lungs  and  the  bronchial  glands. 

TUBERCULOSIS    OF    SWINE. 

The  pig  is  very  susceptible  to  experimental  disease,  and  according 
to  some  authors,  the  scrofulous  conditions  (see  Photograph,  Fig.  53) 
occasionally  observed  in  pigs  are  due  to  tuberculosis.  According  to 
Nocard  the  disease  in  the  pig  often  develops  with  great  rapidity  and 
passes  unperceived.  In  the  chronic  form  the  bacilli  are  rare,  and 
appear  to  have  lost  part  of  their  virulence ;  and  when  inoculated  into 
guinea-pigs  they  produce  a  disease  of  slow  course,  but  the  period  of 
incubation  becomes  shortened  when  the  bacilli  are  inoculated  from 
the  first  guinea-pig  to  others  in  series. 

According  to  Nocard,  nine  out  of  every  ten  pigs  are  infected 
through  the  alimentary  canal.  Many  investigators  have  produced 
the  disease  by  feeding  pigs  with  milk  from  cows  with  tuberculous 
udders.  In  1877,  pigs  were  kept  under  the  shambles  in  San 
Francisco,  and  fed  on  the  offal  which  fell  down  a  shoot.  The 
percentage  of  tuberculosis  that  occurred  in  those  swine  was  beyond 
conception,  the  livers  and  spleen  being  studded  with  masses  of 
tuberculous  nodules.  (For  Photograph  of  a  tuberculous  spleen  of  pig, 
see  Fig.  54,  and  for  Photomicrograph  of  a  section  of  a  tubercle  from 
same,  see  Fig.  52.) 

Tuberculin. — The  use  of  tuberculin  as  a  curative  agent  has  not 
fulfilled  the  expectations  at  first  anticipated.  When  injected  into  healthy 
persons  it  has  no  reaction,  but  in  tubercular  patients  a  pronounced 
systemic  reaction  results.  Koch  has  lately  produced  a  new  tuberculin, 
known  as  TO.  and  TR.,  prepared  by  triturating  dried  cultures  of  tubercle 
bacilli  in  a  mortar  without  anything  being  added  for  a  considerable  time. 
The  mass  is  then  mixed  with  distilled  water  and  placed  in  a  centripetal 
machine,  when  an  opalescent,  transparent,  whitish  fluid  is  obtained  free 
from  bacilli.  Trudeau  and  Baldwin  have  recently  conducted  experiments 
with  this  new  preparation  in  New  York,  and  found  that  it  still  contained 
living  tubercle  bacilli,  capable  of  producing  tuberculosis  in  guinea-pigs ; 

K 


146  SPECIAL  BACTERIOLOGY 

results  since  confirmed  by  other  investigators.  In  preparing  the  original 
tuberculin,  according  to  Kiihne's  investigations,  the  ordinarily  prepared 
culture  media  contain  so  much  peptone  that  there  is  always  more  or 
less  albumose  present,  rendering  it  impossible  to  separate  the  true 
products  of  the  bacillus  from  other  substances  present  in  the  culture 
media,  hence  special  culture  media  formulae  are  used  by  some  investi- 
gators. Koch  in  his  original  method,  after  testing  the  purity  of  six  to 
eight  weeks'  old  veal  bouillon  cultures  of  the  tubercle  bacilli  by 
microscopical  examination,  poured  them  into  a  suitable  vessel,  and 
evaporated  to  one-tenth  the  original  volume  over  a  water-bath.  The 
liquid  was  then  filtered  through  porcelain.  The  crude  tuberculin 
obtained  by  this  process  contains  40  to  50  per  cent,  of  glycerine,  is 
soluble  in  water,  insoluble  in  alcohol,  passes  readily  through  dialyzing 
membranes,  and  is  not  destroyed  by  a  boiling  temperature,  keeps  well, 
and  preserves  its  activity  indefinitely. 

The  original  tuberculin  is  very  valuable  as  a  diagnostic  agent  in  bovine 
tuberculosis.  For  this  purpose  it  is  diluted  with  9  parts  of  water  con- 
taining J  per  cent,  of  carbolic  acid.  About  3  c.c.,  or  60  minims,  are 
injected  subcutaneously ;  the  point  of  injection  is  immaterial,  but  the 
side  of  the  neck  where  the  skin  is  thin  is  the  most  suitable  place.  It  is 
also  more  practical  to  use  a  large-sized  inoculating  needle  instead  of  the 
smaller  varieties ;  the  hair  is  clipped  from  the  part  selected,  which  is 
thoroughly  cleansed  and  disinfected  before  the  injection  is  made.  Before 
the  tuberculin  test  is  applied,  the  temperature  of  the  animals  ought  to 
be  taken  every  two  hours,  at  least  six  or  seven  times  before  the 
injections  are  made,  as  in  many  animals  the  variations  are  sufficiently 
constant  to  make  their  determination  by  precise  measurements  practi- 
cally necessary  in  every  tuberculin  test  from  which  reliable  results  are 
expected.  After  the  injection  is  made  the  temperature  ought  to  be 
taken  again  in  eight  to  ten  hours,  and  from  then  on  every  two  hours, 
until  a  decided  reaction,  continuous  during  several  hours,  has  occurred, 
or  until  eighteen  to  twenty  hours  have  elapsed  since  the  time  of  injection. 
The  febrile  reaction  in  tuberculous  cattle,  following  the  subcutaneous 
injection  of  tuberculin,  begins  six  to  ten  hours  after  the  injection,  reaches 
the  maximum  in  nine  to  fifteen  hours,  and  returns  to  the  normal  in 
eighteen  to  twenty-six  hours  after  the  injection.  The  elevation  in  the 
temperature  sufficient  to  constitute  a  reaction  has  variously  been  given 
from  0*5°  to  1°  C.,  but  consideration  of  the  number  of  degrees  the 
temperature  after  the  injection  rises  above  the  temperature  before  the 
injection  is  not  alone  sufficient — the  height  of  the  temperature  and  the 
duration  of  the  reaction  must  also  be  taken  into  account ;  advanced  cases 
of  tuberculosis  occasionally  fail  to  react,  while  the  reaction  is  frequently 
highest  in  young  animals  in  the  first  stage  of  the  disease.  In  fact,  the 
variation  of  the  temperature  of  an  animal  during  the  course  of  the  day  is 
frequently  so  great  that  if  the  variation  is  not  determined,  and  the 


.VJ.— B.  Tuberculosis.*^  Action  of  tubercle  from  spleen  u(  1> 
'Ehrlich's  method.     X  000. 


FK;.  ,j3.—  Scrofulous  ijtaiid.     Cut  in  two  from  neck  of  \>\£, 
filled  with  caseous  and  calcareous  matter. 


;.  .'i4. — .Spli-en  of  a^  pig,  showing  tubercular  nodules. 

['/'.  lion-hill,  F.R.C.V.X.,  riuili'.,  K<l> nliti r<ih,  1898. 


TUBERCULOSIS   OF   SWINE  147 

temperature  is  taken  only  once  previous  to  a  tuberculin  injection,  it  is 
merely  a  matter  of  chance  if  a  high  temperature,  natural  to  the  animal 
and  independent  of  the  action  of  the  tuberculin  injection,  is  not  con- 
founded with  and  erroneously  taken  for  a  tuberculin  reaction.  The 
range  of  the  thermal  reaction  gives  no  indication  of  the  extent  of  the 
tubercular  lesions  in  the  animal.  Instances  are  recorded  where  a  second 
injection  of  tuberculin  has  altogether  failed  to  produce  a  reaction  in 
animals  which  gave  a  very  decided  reaction  after  the  first  injection,  not- 
withstanding that  the  two  injections  were  separated  by  a  very  consider- 
able period  of  time.  This  question  of  the  non-reaction  to  a  second 
injection  is  a  matter  for  future  investigation.  Should  the  foregoing 
instances  be  correct,  there  is  nothing  to  hinder  unscrupulous  persons  to 
so  prepare  their  animals,  that  when  submitted  to  a  tuberculin  test  the 
results  are  negative,  and  unhealthy  stock  left  as  centres  of  infection. 

Immunity. — At  the  Ninth  International  Congress  of  Hygiene, 
Behring  stated,  '  The  vaccine  of  tuberculosis  has  not  yet  been  found, 
and  that  he  had  hoped  to  find  an  active  antitoxin  with  a  corre- 
sponding toxin,  but  so  far  he  had  been  unsuccessful.  He  would  even 
be  sceptical  as  regards  such  a  result  had  he  not  found  that  birds  were 
much  more  suitable  for  this  kind  of  research  than  mammals.  With 
sodium  and  other  reagents,  mucin  as  well  as  other  chemical  substances 
could  be  extracted  from  tubercle  bacilli.  These  bodies  had  nothing 
to  do  with  the  toxin  of  tuberculosis.  If  the  bacilli  were  subjected 
to  a  temperature  of  105°  C.,  freed  from  fat,  and  treated  with 
glycerinated  water,  insoluble  albuminous  bodies  were  obtained,  which 
had  a  virulence  twenty  times  greater.  The  toxin  of  tuberculosis  was 
therefore  not  identical  with  the  primitive  substance.  The  chemical 
constitution  of  the  toxin  was  not  modified  by  the  operations  to  which 
it  was  subjected.  Behring  stated  the  substance  which  he  had  isolated 
was  eighty  or  even  one  hundred  times  more  virulent  than  Koch's 
tuberculin.  He  had  also  obtained  an  antitoxin  by  passing  the  virus 
through  a  horse  in  the  same  manner  as  was  done  in  the  case  of 
diphtheria  and  tetanus.  There  was,  however,  this  difference,  that  in 
a  phthisical  patient  more  than  0'5  c.c.  to  1  c.c.  could  be  injected 
without  producing  injurious  effects,  general  as  well  as  local.  Behring 
lays  it  down  as  a  principle  that  the  harmlessness  of  an  antitoxin  is  an 
indispensable  condition  of  its  practical  application,  but  there  are 
many  other  difficulties  which  must  be  overcome  before  an  antitoxin 
of  tuberculosis  can  come  into  use  in  the  treatment  of  human  beings. 
In  bovine  animals  it  is  already  possible  by  its  means  to  cure  a 
declared  tuberculosis,  but  even  in  them  one  gathers  that  the  remedy 
at  present  is  somewhat  dangerous  to  life.  This,  however,  as  pointed 


U8  SPECIAL  BACTERIOLOGY 

out  by  Behring,  is  important  only  from  an  economic  point  of  view. 
If  of  the  one  hundred  animals  treated  with  the  serum  ninety  are  cured 
and  ten  are  killed,  the  treatment  should  still  be  adopted.  Experiments 
to  determine  the  value  of  this  treatment  are  to  be  conducted  on  an 
extensive  scale  in  the  Berlin  Veterinary  School. 


AVIAN   TUBERCULOSIS. 

The  bacillus  of  avian  tuberculosis  is  closely  allied  to  the  bacillus 
of  mammalian  tuberculosis. 

Microscopical  Appearances. — It  is  thinner  and  more  slender  than 
the  bacilli  found  in  man  and  mammals,  and  club  and  branched  forms 
are  more  frequent  (see  Photomicrograph,  Fig.  55). 

Staining  Reactions.— It  is  easier  stained  than  the  bacilli  of  man 
and  mammals,  but  exhibits  a  similar  reaction  towards  decolorizing 
agents. 

Biological  Characters. — It  is  not  so  difficult  to  cultivate  as  the 
other  forms,  growing  in  ordinary  agar  and  in  ordinary  bouillon ;  but  the 
addition  of  glycerine  to  the  media  assists  the  growth  to  a  great  extent, 
which,  moreover,  is  much  quicker  than  the  growth  of  the  bacillus  of 
mammalian  tuberculosis.  The  cultures  are  not  so  dry,  but  much  moister, 
forming  a  coherent  coating ;  and  on  solid  culture  media  the  growth 
forms  a  film  over  the  water  of  condensation.  Old  cultures  exhibit  a 
yellowish  colour. 

Differential  Characteristics. — The  bacillus  of  avian  tuberculosis 
grows  just  as  luxuriantly  at  42,  43,  45°  C.  as  at  37°  C.,  a  characteristic  not 
exhibited  by  the  bacillus  of  human  tuberculosis,  as  it  ceases  to  develop 
at  this  high  temperature.  Supposing  both  forms  to  be  identical  species, 
the  effects  of  the  high  temperature  on  the  cultures  may  be  due  to  the 
avian  bacilli  having  become  adapted  to  a  high  temperature  during  their 
sojourn  in  the  body  of  the  bird,  the  normal  temperature  of  same  being 
41°  to  42°  C.  The  bacilli  of  avian  tuberculosis  are  more  resistant  towards 
heat  than  the  bacilli  of  human  tuberculosis,  being  first  killed  by  exposure 
for  fifteen  minutes  at  70°  C. 

The  bacilli  are  found  in  the  tuberculous  lesions,  which  are  charac- 
terised by  tough  masses  of  nodules,  calcareous  infiltration  often  occurring. 
Giant  cells  are  very  scarce.  A  few  cases  of  avian  tuberculosis  have  been 
observed  in  man  and  mammals. 

Pathogenesis. — The  most  of  birds  are  very  susceptible,  and  can  be 
infected  by  all  the  different  methods  of  infection.  According  to  Baum- 
garten,  the  spontaneous  outbreaks  are  in  most  cases  congenital.  Guinea- 
pigs  and  dogs  are  somewhat  refractory,  without,  however,  possessing  a 


AVIAN  TUBERCULOSIS  149 

perfect  immunity.  The  avian  tuberculous  bacilli  usually  develop  badly  in 
mammals ;  and,  on  the  other  hand,  mammalian  tubercle  bacilli  are 
acclimated  with  difficulty  in  birds. 

At  the  recent  Congress  in  Paris,  M.  Nocard  communicated  the 
following  interesting  facts  regarding  avian  tuberculosis.  The  fowl 
cannot  be  infected  by  inoculation  with  tuberculosis  from  a  human  being 
any  more  than  from  a  bovine.  On  the  other  hand,  the  dog  and  guinea- 
pig,  although  most  susceptible  to  the  action  of  both  human  and  bovine 
tuberculosis,  are  very  refractory  to  avian  tuberculosis.  If,  however,  a 
guinea-pig  is  inoculated  intraperitoneally  with  avian  tuberculous  material, 
it  often  dies,  showing  a  special  kind  of  lesion.  The  sputum  of  a 
tuberculous  patient  will  kill  a  rabbit  by  inoculation,  but  only  very  rarely 
a  guinea-pig.  M.  Nocard  enveloped  a  glycerinated  bouillon  culture  of 
human  tuberculosis  in  little  sacs  of  collodion,  and  placed  them  in  the 
peritoneal  cavities  of  poultry.  The  sacs  were  removed  in  from  five  to 
eight  months,  and  found  to  contain  a  sort  of  paste  made  up  of  bacilli. 
Cultures  instituted  from  this  paste  grew  extremely  well,  and  the 
interesting  fact  was  observed  that  the  bacillus  had  lost  its  human 
characteristics  and  assumed  those  of  the  avian  bacillus.  It  grew  at  high 
temperatures,  and  the  cultures  did  not  present  the  characteristic  frayed- 
out  lumps.  The  bacillus  was  not  virulent  enough  to  produce  tuberculosis 
in  fowls  until  it  was  passed  through  two  or  three  fowls,  and  a  period  of 
four  to  six  months  had  elapsed.  In  one  case  a  fowl  suddenly  contracted 
tuberculosis  eleven  months  after  the  introduction  of  the  collodion  sac. 
At  the  post-mortem  examination  it  was  found  that  the  sac  had  burst, 
showing  that  development  had  proceeded  far  enough  when  the  sac 
ruptured  to  produce  tubercle  in  the  fowl.  Nocard  considers  that  human 
and  avian  tuberculosis  are  only  two  different  varieties  of  the  same  disease. 

Diagnosis. — The  examination  of  the  tuberculous  material  for 
bacilli  is  conducted  the  same  as  the  examination  of  human  and 
mammalian  material. 

Nocard  states  that  if,  as  appears  probable  from  his  investiga- 
tions with  equine  tuberculosis,  the  human  subject  may  contract 
tuberculosis  from  the  fowl,  the  most  elementary  prudence  requires 
that  the  sale  of  fowls  coming  from  a  place  in  which  the  disease  exists 
ought  to  be  interdicted,  for  the  consumption  of  a  fowl  in  the  roasted 
condition  involves  the  risk  of  the  ingestion  of  a  considerable  number 
of  living  and  virulent  bacilli. 


PSEUDO-TUBERCULOSIS. 

The  term  '  pseudo-tuberculosis '  is  applied  to  certain  pathological 
processes  which  resemble  the  genuine  tubercle,  but  are  dependant  on 


150  SPECIAL  BACTERIOLOGY 

other  causes  other  than  Koch's  bacillus  of  tuberculosis.  The  etiology 
of  pseudo-tuberculosis  is  remarkably  manifold.  The  following  are 
known  as  causal  factors  of  pseudo-tuberculosis  :— 

1.  Inanimate  foreign  bodies. 

2.  Animal  parasites. 

3.  Bacteria. 

4.  Vegetable  parasites. 

These  pseudo-tuberculoses  caused  by  foreign  bodies  can  be  pro- 
duced easily  with  all  kinds  of  substances,  but  are  not  transmissible 
from  animal  to  animal. 

The  pseudo- tuberculosis  produced  by  animal  parasites  is  only 
found  in  the  lower  animals.  In  the  cat  it  is  caused  by  the  Ollulanus 
tricuspis ;  in  the  sheep  by  the  Pseudalms  ovis  pulmonalis  ;  in  the  calf 
by  the  Strongylus  refuscens  ;  in  the  dog  by  the  Strongylus  vasorum. 

Muira  records  the  only  case  in  man,  which  occurred  in  a  patient 
that  died  of  beri-beri,  a  fibrous  tubercle  being  found  in  the  mesentery 
caused  by  distoma  eggs. 

Many  cases  of  pseudo-tuberculosis  in  animals  caused  by  bacteria 
are  mentioned,  the  principal  being  zooglceic  tuberculosis,  described 
by  Malassez  and  Vignal. 

Microscopical  Appearances.  — -  Thick,  short  rods,  frequently 
exhibiting  cocci  in  the  form  of  chains  in  small  groups  or  in  zooglcea. 
Spore  formation  is  absent. 

Staining  Reactions. — The  reaction  with  the  Gram  method  is 
negative.  Sections  are  best  stained  with  Malassez's  blue  prepared  as 
follows : — 

Two  per  cent,  solution  of  carbonate  of  soda,     -  10  c.c. 

Saturated  anilin  water,  5  c.c. 

Absolute  alcohol,  3  c.c. 

Solution  made  with  9  vols.  of  distilled   water  and   1   vol.  'j 

of  concentrated  solution  of  methylene  blue  in  90  per  >   3  c.c. 
cent,  alcohol, 

Sections  remain  in  this  solution  two  or  three  days,  and  are  then 
washed  in  water  stained  with  methylene  blue,  and  cleared  in  oil  of 
bergamot  or  turpentine. 

Biological  Characters.  —  On  Gelatine  Plates  colonies  develop 
somewhat  similar  to  those  of  typhus  abdominalis.  The  medium  is 
not  liquefied. 

In  Gelatine  Stab  Cultures  the  growth  resembles  a  flat  nail. 

On  Agar,  a  greyish,  foetid  growth. 


PSEUDO-TUBERCULOSIS  151 

On  Potatoes,  a  yellowish  coating. 

In  Bouillon  a  flaky  cloudiness  occurs  at  first,  then  a  sediment  is 
formed,  the  upper  portions  of  the  medium  becoming  clear. 

Pathogenesis. — The  bacillus  of  pseudo-tuberculosis  is  pathogenic 
for  guinea-pigs,  death  occurring  in  five  to  six  days,  also  for  dogs  and 
horses.  The  post-mortem  lesions  resemble  those  of  genuine  (Koch) 
tuberculosis,  especially  in  the  abdominal  organs,  which  the  pseudo- 
bacillus  especially  attacks.  The  differential  diagnosis  is,  however,  not 
difficult,  the  easy  staining  and  the  quick  growth  of  the  pseudo-bacillus 
yielding  a  distinction  without  difficulty. 

Courmont  also  describes  a  bacillus  found  in  tubercular  lesions  of  the 
pleura  of  the  ox  (the  bacilli  of  Koch  being  absent).  This  bacillus  is 
short,  with  its  substance  condensed  at  both  ends,  and  a  clear,  slightly 
constricted  middle  ;  it  does  not  form  chains  or  diplococci.  It  is  both 
aerobic  and  anaerobic,  grows  quickly,  is  easily  cultivated  in  all  kinds  of 
media  up  to  46°  C.  It  is  pathogenic  for  guinea-pigs,  which  die  in  four 
to  eight  days  with  generalized  tuberculosis,  and  for  rabbits,  in  which 
disseminated  and  confluent  tubercles  are  found  in  the  spleen,  liver,  and 
lungs.  The  bacillus  is  also  found  in  the  blood  of  inoculated  animals, 
and  it  also  becomes  generalized  without  affecting  the  lymphatic  glands. 
The  pseudo-tuberculosis  caused  by  the  higher  organized  vegetable 
parasites  likewise  manifests  itself,  especially  in  animals.  Different  forms 
of  streptothrix  and  aspergilli,  particularly  the  Aspergillus  glaucus  and 
fumigatus,  require  consideration.  Pigeons  often  succumb  from  a 
miliary  tuberculosis,  the  Aspergillus  fumigatus  being  found  present  in 
the  interior  of  the  granulations.  Lung  affections  are  also  sometimes 
observed  in  individuals  engaged  in  the  feeding  of  pigeons,  which  are 
apparently  due  to  the  same  parasite — at  least  the  Aspergillus  fumigatus 
is  found  in  the  expectoration  of  these  patients.  It  is  probable  that  the 
parasite  is  conveyed  writh  the  grain  used  to  feed  the  pigeons.  Eppinger 
records  a  case  of  pseudo-tuberculosis  in  man  due  to  the  Streptothrix 
Eppinger,  see  page  108. 

Valee  has  recently,  in  the  Recueil  de  Medecine  ffeterinaire,  described  a 
new  form  of  pseudo-tuberculosis  in  calves  due  to  an  organism  which  is 
much  smaller  than  that  of  swine  erysipelas.  It  occurs  either  isolated  or 
in  small  masses  in  the  diseased  tissues,  and  stains  by  the  Gram-Nicolle 
process.  When  isolated  and  cultivated  on  the  different  media,  it  pro- 
duces the  original  lesions  in  the  various  animals  experimented  upon. 
The  principal  lesions  in  the  affected  calves  are  in  the  liver,  which  is 
normal  in  size,  but  covered  with  fine  granulations  or  tubercles  of  a 
greyish  colour,  in  some  cases  confluent.  There  was  also  a  slight  peri- 
hepatitis,  and  the  tissue  of  the  gland  was  very  friable. 


152  SPECIAL  BACTERIOLOGY 


BACILLUS    LEPR.E. 

This  organism,  discovered  by  Hansen  in  1879,  is  found  chiefly  in 
the  interior  of  the  peculiar  round  and  oval  cells  found  in  leprous 
tubercles.  The  bacilli  have  also  been  found  in  the  lymphatic  glands, 
liver,  spleen,  testicles,  and  in  the  thickened  portions  of  nerves 
involved  in  the  anaesthetic  form  of  the  disease.  According  to  some 
authorities  they  have  also  been  found  in  the  blood.  The  bacilli  lie  in 
the  leprous  cells  in  great  numbers,  and  also  in  the  lymph  spaces  out- 
side of  these  cells.  They  are  not  found  in  the  epidermal  layers  of  the 
skin,  but,  according  to  Babes,  they  may  penetrate  the  hair  follicles. 

Microscopical  Appearances. — The  Bacillus  leprae  resembles  the 
tubercle  bacilli  in  form,  but  is  more  uniform  in  length  and  not  so 
frequently  bent  or  curved,  and  is  from  4  to  6  /u,  in  length,  and  less  than 
1  p  in  width  ;  the  ends  of  the  rods  are  pointed,  and  in  stained  specimens 
unstained  spaces  similar  to  those  in  the  Bacillus  tuberculosis  are  present. 

Motility.— Non-motile. 

Staining  Reactions. — The  bacilli  stain  readily  with  the  aniline 
dyes,  also  by  the  Gram  method  and  by  the  Cladius  method  (see  Photo- 
micrograph, Plate  III.,  Fig.  17).  For  differential  staining  reactions  see 
Tuberculosis,  p.  136. 

Biological  Characters.— It  has  not  yet  been  obtained  in  pure 
cultures,  so  that  its  etiological  relation  to  the  disease  with  which  it  is 
associated  is  based  upon  the  demonstration  of  its  constant  presence  in 
leprous  tissues. 

Pathogenesis.  —  Leprous  tissues  containing  the  bacillus  are  in- 
fectious, and  may  produce  this  disease.  Arning  inoculated  a  condemned 
criminal  in  the  Sandwich  Islands  subcutaneously  with  fresh  leprosy 
tubercles,  who  was  under  observation  until  his  death  occurred  from 
leprosy  at  the  end  of  five  years.  Positive  results  have  also  been  obtained 
in  the  lower  animals.  Melcher  and  Ortmari  inoculated  rabbits  with 
pieces  of  fresh  leprosy  tubercles  in  the  anterior  chamber  of  the  eye,  the 
animals  dying  at  the  end  of  several  months ;  the  characteristic  tubercles 
containing  the  bacilli  being  found  distributed  throughout  the  various 
organs. 


BACILLUS  SMEGMATIS. 

Found  in  the  smegma  praeputii  between  the  scrotum  and  thigh 
and  between  the  labiae.  It  is  also  found  in  the  cerumen,  and  occasion- 
ally on  the  skin. 


Fu).  55.— Bacillus  of  Avian  Tuberculosis.     'Branched   forms.'     Cover-glass 
specimen  from  the  liver  of  a  chicken.    Ehrlich's  method,    x  1000. 


<"¥*;• 


FIG.  5G.—B.  Cholene  Asiatic*.     Agar  culture.     Fuchsin.     X  1000. 
\T.  tloirlriU,  F.I'.C.r.S.,  Photo., 


THE  COMMA  BACILLUS  OF  ASIATIC  CHOLERA         153 

Microscopical  Appearances. — The  bacilli  lie  in  clusters  either  in 
or  between  the  epithelial  cells,  the  size  and  form  of  the  rods  being  very 
similar  to  the  Bacillus  tuberculosis. 

Staining  Reactions. — They  stain  with  difficulty,  are  acid-resisting 
when  stained  by  the  methods  for  tubercle  bacilli  (see  Technique,  §§13 
and  14),  but  are  decolorized  when  treated  one  minute  with  absolute 
alcohol.  The  reaction  with  the  Gram  method  is  positive. 

Biological  Characters. — Doutrelepont  and  Mattel-stock  obtained  a 
culture  of  a  similar  organism  on  coagulated  hydrocele  fluid,  which 
coloured  the  medium  brown ;  further  cultivation  was  not  successful. 

Differential  Diagnosis  (see  Bacillus  Tuberculosis,  p.  137). — It  is 
most  likely  to  be  mistaken  for  the  Bacillus  tuberculosis  in  the  examina- 
tion of  urine. 

THE   COMMA   BACILLUS   OF   ASIATIC   CHOLERA. 

(Vibrio  Cholerae  Asiatic*.) 

In  1883  Koch  discovered  that  in  all  cases  of  Asiatic  cholera  a 
particular  form  of  bacterium  was  present,  and  that  these  bacteria  were 
found  exclusively  in  cases  of  genuine  Asiatic  cholera. 

Microscopical  Appearances. — Slightly  curved  rods  with  rounded 
ends  from  0'8  to  2  //,  in  length,  and  about  0*3  to  0'4  /x,  in  breadth. 
The  rods  are  usually  curved  like  a  comma,  but  are  occasionally  in  the 
form  of  a  half  circle,  or  two  contact  rods  curved  in  opposite  directions 
may  form  an  S-shaped  figure.  The  typical  comma  shape  is  best  observed 
in  specimens  prepared  from  young  cultures  (see  Photomicrograph,  Fig.  56). 
When  newly-developed  individual  bacilli  remain  attached  together,  they 
form  long  spirals.  This  condition  is  frequently  observed  in  cultures,  espe- 
cially in  old  cultures  or  on  the  addition  of  weak  antiseptics,  i.e.  alcohol. 
In  human  cholera  dejections,  the  spirillum  forms  are  extremely  seldom 
observed.  In  the  peritoneal  exudate  of  inoculated  guinea-pigs  spirillum 
forms  are  especially  frequent.  The  spirilla  are  considered  to  be  involution 
forms. 

Motility. — Strongly  motile.  When  examined  in  a  hanging-drop  cul- 
ture they  resemble  a  swarm  of  gnats.  The  motility  is  due  to  one 
flagellum  separated  at  the  end  of  the  rod  (Monotricha).  (See  Photomicro- 
graph, Plate  II.,  Fig.  10,  stained  by  the  author's  orcein  method). 

Spore  Formation. — Spores  do  not  exist.  The  arthrospores  described 
by  Hueppe  have  not  been  confirmed  by  other  investigators. 

Staining  Reactions. — The  best  results  are  obtained  with  a  satu- 
rated watery  solution  of  fuchsin  or  with  carbol  fuchsin.  The  stain  must 


154  SPECIAL  BACTERIOLOGY 

be  allowed  to  work  a  considerable  time.  The  reaction  with  Gram's 
method  is  negative.  Sections  may  be  stained  with  Loffler's  methyleiie 
blue  solution. 

Biological  Characters. — The  comma  bacillus  grows  on  all  the 
ordinary  nutrient  media,  also  in  the  absence  of  oxygen  (facultative 
anaerobe),  but  according  to  recent  investigations  the  oxygen  must  not 
be  completely  absent.  The  culture  media  must  possess  a  decided  alkaline 
reaction,  as  the  comma  bacillus  is  very  sensitive  to  the  smallest  quantity 
of  acid. 

The  minimum  temperature  at  which  development  takes  place  is 
8°  C.,  optimum  30°  to  40°  C. 

On  Gelatine  Plates  the  development  takes  place  best  at  22°  C.,  and 
in  twenty-four  to  thirty  hours,  when  examined  under  a  low  power,  small 
yellowish-white  granular  knotty  colonies  with  uneven  rough  edges  are 
present,  the  surface  looks  as  if  it  were  covered  with  little  fragments  of 
broken  glass  ;  while  the  colony  has  a  shining  appearance  when  lique- 
faction commences.  An  ill-defined  halo  is  first  seen  to  surround  the 
granular  colony,  which  exhibits  a  peculiar  roseate  hue  by  transmitted 
light. 

In  Gelatine  Stab  Cultures  development  occurs  along  the  line  of  inocula- 
tion, but  the  liquefaction  occurs  first  only  at  the  surface ;  on  the  second 
day,  at  22°  C.,  a  short  funnel  is  formed  with  a  very  narrow  mouth,  the 
upper  portion  of  which  contains  air,  and  below  this  a  whitish  viscid  mass. 
The  funnel  now  increases  in  depth  and  diameter,  and  in  four  to  six  days 
may  reach  the  edge  of  the  test-tube  ;  in  eight  to  fourteen  days  the  upper 
two-thirds  of  the  gelatine  is  liquefied,  and  in  a  few  weeks  the  gelatine 
is  completely  liquefied. 

On  Agar  Plates  the  growth  is  not  so  characteristic  as  with  gelatine 
media ;  the  surface  colonies  exhibit  a  peculiar  light  greyish-brown  trans- 
parent appearance. 

On  Agar  Stroke  Cultures  a  greyish-white,  moist,  shining  coating 
develops. 

Blood  Serum  is  liquefied  slowly. 

On  Potatoes  placed  in  the  incubator  a  thin  semi-transparent  brown  or 
greyish-brown  layer  is  developed.  In  some  potatoes  no  growth  takes 
place,  but  development  takes  place  if  the  potatoes  are  rendered  alkaline 
with  a  solution  of  soda,  or  cooked  in  a  3  per  cent,  solution  of  common 
salt. 

Milk  is  a  favourable  culture  medium. 

Bouillon  is  clouded,  and  in  the  majority  of  cases,  when  placed  in  the 
incubator,  a  thin  membrane  forms  on  the  surface  of  the  medium. 

Like  most  vibrios  and  spirilla  the  comma  bacillus  possesses  the 
peculiarity  of  being  able  to  multiply  very  energetically  in  bouillon 
reduced  with  six  to  eight  parts  of  water.  It  also  develops  in  1  per  cent. 


THE  COMMA  BACILLUS  OF  ASIATIC  CHOLERA         155 

watery  solution  of  peptone,  to  which  ^  per  cent,  of  chloride  of  sodium  is 
added,  and  if  the  peptone  used  is  not  alkaline,  then  the  medium  must  be 
rendered  alkaline  with  a  solution  of  soda. 

Specific  Reactions. — When  a  drop  of  pure  reduced  hydrochloric 
or  sulphuric  acid  is  added  to  cultures  of  cholera  bacilli  grown  in  peptone 
media,  a  rose  or  purple-red  colour  results,  which  is  known  as  the 
nitroso-indol  reaction.  The  comma  bacilli  possess  the  faculty  of  first 
forming  indol,  and  then  changing  the  traces  of  nitrates  in  the  culture 
solution  into  nitrites.  Other  vibrios  exhibit  the  nitroso-indol  reaction 
besides  the  cholera  vibrio,  i.e.,  vibrio  Mentschnikoff — vibrio  Berolinesis 
(see  section  on  Water  Bacteria,  page  206).  Finkler  and  Prior's  vibrio  and 
Denecker's  cheese  vibrio  also  form  indol,  but  no  nitrite,  the  addition  of 
a  pure  acid  that  does  not  contain  nitrous  acid  producing  no  red  colour. 
In  bouillon,  under  certain  circumstances,  the  reaction  fails  when  either 
too  much  or  too  little  nitrates  are  present. 

There  are  two  methods  whereby  genuine  cholera  bacilli  are 
differentiated  from  other  similar  vibrios,  known  as  Pfeiffer  and  Gruber's 
reactions. 

(1.)  Pfeiffer 's  Reaction  is  produced  as  follows  : — Some  blood  serum  of 
a  guinea-pig  or  other  animal  rendered  immune  to  cholera  is  reduced 
with  ordinary  bouillon  in  the  proportion  of  1  to  100,  and  in  1  c.c.  of 
the  above  mixture,  a- platinum  loop  (capable  of  holding  about  2  mg.), 
full  of  the  vibrio  species  under  investigation,  is  added,  and  the  inoculated 
mixture  injected  into  the  peritoneal  cavity  of  a  guinea-pig  weighing  about 
200  grams.  Every  five  minutes  some  of  the  peritoneal  effusion  forming 
is  removed  by  means  of  fine  glass  capillary  pipettes,  and  examined, 
both  stained  and  unstained.  If  it  is  the  genuine  Koch's  comma  bacillus, 
they  will  be  observed  to  become  non-motile  first,  then  transform  them- 
selves into  small  balls,  which  finally,  in  about  twenty  minutes,  become 
quite  loosened.  When  the  above  phenomena  are  absent,  then  the 
vibrio  belongs  to  another  species.  To  prevent  an  error  arising,  a 
control  guinea-pig  is  inoculated  iiitraperitorieally  with  1  c.c.  of  normal 
serum  bouillon  mixture,  1  to  100,  to  which  one  loop  of  the  suspected 
culture  is  added.  If  in  twenty  minutes  the  peritoneal  effusion  contains 
living  motile  bacilli  (which  were  killed  with  immune  serum),  then  the 
diagnosis  of  Asiatic  cholera  can  be  given  with  safety. 

(2.)  Gruber's  Reaction. — A  small  portion  of  the  vibrio  culture  under 
investigation  is  placed  with  the  serum  of  an  animal  vaccinated  against 
cholera  in  the  proportions  of  1  to  50,  1  to  100,  and  upwards.  The  mix- 
ture is  examined  at  once  with  a  high  power.  If  the  vibrio  become 
non-motile,  flock  together  in  herds,  and  agglutinate,  then  the  bacilli 
are  without  doubt  genuine  cholera  vibrio. 

A  macroscopic  agglutination  can  also  be  procured  by  inoculating 
bouillon  with  the  suspected  vibrio  and  adding  cholera-immune  serum  in 


156  SPECIAL  BACTERIOLOGY 

the  same  proportions  as  above.  If  in  sixteen  to  twenty-four  hours  the 
vibrioiies  are  rolled  together  in  flakes  at  the  bottom  of  the  reagent 
glass,  and  the  upper  portions  of  the  fluid  remain  clear,  then  the 
reaction  is  the  same  as  that  exhibited  by  the  genuine  cholera  vibrio. 

Vitality. — The  comma  bacilli  do  not  exhibit  much  resistance.  They 
are  destroyed  in  water  heated  to  52°  C.  in  four  minutes.  They  withstand 
lower  temperatures  better ;  in  ice  they  lose  their  vitality  in  a  few  days. 
The  addition  of  0'07  to  0'08  per  cent,  hydrochloric  or  nitric  acid  to 
neutral  culture  media  prevents  their  growth ;  this  explains  why  normal 
gastric  juice,  which  contains  about  0'2  per  cent,  of  hydrochloric  acid, 
exhibits  an  insurmountable  obstacle  to  the  cholera  bacilli.  When  spread 
in  a  thin  layer  and  dried  they  lose  the  faculty  of  further  development  in 
three  hours.  In  moist  surroundings  under  favourable  circumstances 
Koch's  bacilli  live  a  long  time — about  nine  months.  In  six  months  old 
agar  and  gelatine  cultures  they  sometimes  still  retain  their  vitality. 
Weak  solutions  of  the  ordinary  antiseptics  kill  the  cholera  bacillus  very 
quickly ;  a  J  per  cent,  solution  of  carbolic  acid  kills  them  in  a  few 
minutes.  In  the  dejections  of  cholera  patients  the  bacilli  sometimes 
remain  alive  for  weeks,  but  this  occurs  only  under  extremely  favourable 
circumstances.  The  cholera  bacillus  multiplies  to  some  extent  in 
sterilized  river  or  well  water,  preserving  its  vitality  in  such  water  for 
several  months.  In  milk  and  water  containing  other  bacteria  it  dies  out 
in  a  few  days.  In  greatly  diluted  bouillon  media  the  cholera  bacilli  may 
take  the  precedence  of  the  common  saprophytic  bacteria,  forming  upon 
the  surface  of  the  medium  the  characteristic  film. 

The  comma  bacilli  only  survive  for  a  few  days  when  mixed  with 
normal  faeces. 

Pathogenesis. — The  introduction  of  cholera  bacilli  into  the  stomach 
of  man  may  cause  110  bad  result,  but  sometimes  a  more  or  less  intensive 
diarrhoea  results  (self-infection  by  Pettenkoifer  and  Emmerich),  and  in 
other  cases  genuine  dangerous  cholera  with  all  the  clinical  symptoms. 
A  young  doctor  died  in  Hamburg  of  typical  cholera,  caused  by  a  drop  of 
peritoneal  exudate  containing  vibrio  getting  into  his  mouth  during  the 
demonstration  of  PfeifFer's  reaction.  Subcutaneous  injection  with  cholera 
bacilli  in  man  only  causes  local  symptoms  and  slight  fever.  According 
to  Klemperer  the  blood  thereby  acquires  immunizing  properties. 

A  disease  resembling  cholera  can  be  produced  in  guinea-pigs  by 
direct  introduction  of  the  vibrio  into  the  duodenum — evading  the  stomach 
and  tying  the  gall-duct— or  by  introduction  of  the  bacilli  into  the 
stomach,  previously  rendered  alkaline  with  a  solution  of  soda,  and  at  the 
same  time  injecting  2  to  3  c.c.  of  tincture  of  opium  into  the  peritoneal 
cavity.  Tying  the  gall-duct  and  injecting  tincture  of  opium  into  the 
peritoneal  cavity  interrupts  the  peristaltic  action  of  the  bowels. 

Bacteriological   Diagnosis.— (1.)  Microscopical   Examination.— 


THE  COMMA  BACILLUS  OF  ASIATIC  CHOLERA         157 

Cover-glass  specimens  are  prepared  from  the  mucus  in  the  faeces, 
stained  with  a  reduced  solution  of  carbol  fuchsin.  The  diagnosis  of 
Asiatic  cholera  is  very  probable  when  the  individual  bacilli  appear  to 
lie  behind  each  other  in  a  direction  like  a  small  swarm  of  fish  in  a 
slowly  running  stream,  but,  nevertheless,  culture  experiments  must 
be  instituted. 

(2.)  Examination  by  Cultures.  —  Gelatine  plate  cultures  are 
instituted  from  the  faeces,  when  possible  from  a  flake  of  mucus.  The 
usual  reductions  are  made  (see  Technique,  §  108)  for  the  special 
characteristic  growth  on  gelatine  of  the  colonies  of  Koch's  bacillus 
(see  Biological  Characters,  p.  154). 

(3.)  The  Peptone  Water  Culture  Method  of  Koch  and  Schotellius. 
— Besides  the  institution  of  plate  cultures,  peptone  water  cultures 
must  be  prepared,  because  in  cases  where  the  cholera  bacilli  are  not 
numerous  they  do  not  develop,  being  overcome  by  the  ordinary  faeces 
bacteria. 

An  Erlenmeyer  flask  containing  1  per  cent,  peptone  and  J  per 
cent,  common  salt  solution  is  inoculated  with  a  platinum  loop  of  the 
suspected  faeces  or  mucus  and  placed  in  the  incubator  at  37°  C.  As 
soon  as  the  fluid  exhibits  the  slightest  trace  of  turbidity,  which 
generally  occurs  in  six  to  ten  or  twelve  hours,  a  portion  is  removed 
from  the  surface  and  examined  in  a  hanging-drop,  and  cover-glass 
specimens  prepared.  If  a  pure  culture  is  obtained,  then  the  diagnosis 
is  assured.  It  is  not  always  so  simple,  as  the  surface  growth  is  some- 
times contaminated  with  other  bacteria,  most  frequently  the  Bacterium 
coli  commune,  and  it  is  therefore  necessary  to  make  plate  cultures, 
and  by  this  means  isolate  the  Koch  bacillus,  which  has  now  increased 
in  the  peptone  solution,  and  numerous  colonies  develop  in  the  Petri- 
dishes. 

Pure  cultures  are  now  prepared  from  the  plate  cultures,  and  tested 
by  the  nitroso-indol  reaction,  as  well  as  by  the  previously  mentioned 
Gruber  and  PfeifFer  reaction.  Animal  experiments  are  also  instituted. 
Should  all  these  tests  yield  positive  results,  then  the  diagnosis  is  con- 
clusive and  certain.  Agar  plates  can  be  used  instead  of  gelatine 
plates,  and  possess  the  advantage  that  they  can  be  placed  in  the 
incubator  at  37°  C.  and  examined  in  eight  to  ten  hours. 

THE  EXAMINATION  OF  WATER  FOR  CHOLERA 
BACILLI. 

It  is  necessary,  to  obtain  satisfactory  results,  to  use  enormous 
quantities  of  the  suspected  water.  About  100  to  1000  c.c.  of  the 


158  SPECIAL  BACTERIOLOGY 

suspected  water  is  placed  in  sterile  flasks,  and  to  each  sample  1  per  cent, 
alkali  peptone  (Witte's  peptone  is  the  best)  and  £  per  cent,  of  common 
salt  is  added.  The  peptone  and  salt  are  kept  ready  for  use  in  sterile 
solutions.  The  mixture,  after  its  alkalinity  is  tested,  is  placed  in  the 
incubator  and  examined  and  tested  in  the  same  manner  as  the  previously 
mentioned  peptone  water -culture  method  of  Koch  and  Schotellius. 


SPIRILLUM   OF   FINKLER   AND   PRIOR. 

(Vibrio   Proteus.) 

This  vibrio  was  isolated  from  the  dejections  of  patients  with 
cholera  nostras  which  had  been  allowed  to  stand  for  some  days,  but 
it  has  since  been  proven  to  possess  no  etiological  significance  in  that 
disease.  To-day  it  is  only  of  historical  interest,  the  cultures  being 
transferred  from  tube  to  tube  in  the  laboratory. 

Microscopical  Appearances. — It  bears  a  great  resemblance  to 
the  vibrio  of  the  Asiatic  cholera,  but  the  curved  segments  are  somewhat 
longer  and  thicker,  and  not  so  uniform  in  diameter,  being  often  thicker 
in  the  middle  than  at  the  poles.  The  spiral  filaments  are  not  so 
numerous,  and  also  shorter  than  those  formed  by  the  cholera  vibrio. 
In  unfavourable  media  involution  forms  are  common. 

Motility. — Strongly  motile,  possesses  a  single  flagellum  at  one  end 
(Monotricha).  (See  Photomicrograph,  Plate  II.,  Fig.  9,  stained  by  the 
author's  orcein  method). 

Staining  Reactions. — Stains  with  the  ordinary  aniline  dyes,  best 
with  an  aqueous  solution  of  fuchsin. 

Biological  Characters. — Aerobic  and  facultative  anaerobic  lique- 
fying vibrio  ;  grows  at  ordinary  room  temperature. 

On  Gelatine  Plates  small  white  punctiform  colonies  develop  in  twenty- 
four  hours,  which  under  microscopic  examination  are  seen  to  be  finelv 
granular  and  yellowish  or  yellowish-brown  in  colour  ;  the  gelatine  around 
the  colonies  liquefies  rapidly,  and  when  the  organism  is  abundant,  lique- 
faction is  usually  complete  in  twenty-four  hours.  Isolated  colonies  on 
the  second  day  form  saucer-shaped  depressions  in  the  gelatine. 

In  Stab  Cultures  liquefaction  progresses  much  more  rapidly  than  with 
the  cholera  vibrio,  a  stocking-shaped  pouch  of  liquefied  gelatine  appearing 
in  two  days,  the  whole  medium  being  liquefied  in  about  a  week  ;  a  whitish 
film  forms  on  the  surface  of  the  liquefied  medium. 

On  Agar  Media  a  moist  shiny  layer  covering  the  entire  surface  is 
quickly  developed. 

On  Blood  Serum  the  growth  is  rapid  and  causes  liquefaction  of  the 
medium. 


SPIRILLUM  TYROGENUM  159 

On  Potato  at  room  temperature  a  shiny,  greyish-yellow,  glistening  layer 
occurs,  soon  spreading  over  the  surface  of  the  potato.  (The  vibrio 
of  Asiatic  cholera  exhibits  no  growth  on  potato  at  room  temperature.)  The 
cultures  give  off  a  strong  putrefactive  odour,  and  in  media  containing 
sugar  produce  an  acid  reaction. 

Pathogenesis. — Pathogenic  for  guinea-pigs  when  injected  into  the 
stomach  previously  rendered  alkaline  with  soda. 

SPIRILLUM  TYROGENUM. 

(Vibrio  Deneke.) 

This  vibrio  was  obtained  by  Deneke  from  old  cheese. 

Microscopical  Appearances. — Curved  rods  and  long  spiral  fila- 
ments resembling  the  cholera  vibrio,  the  diameter  of  the  commas  being 
uniform  throughout,  so  that  it  more  closely  resembles  the  cholera  vibrio 
than  does  that  of  Finkler  and  Prior. 

Motility. — Strongly  motile,  possessing  a  single  flagellum  at  one  end 
(Monotricha). 

Staining  Reactions. — Stains  with  the  usual  aniline  dyes,  best  with 
an  aqueous  solution  of  fuchsin. 

Biological  Characters. — Aerobic  and  facultative  anaerobic  lique- 
fying vibrio,  growing  at  ordinary  room  temperature  more  rapid  than  the 
cholera  vibrio,  but  not  so  rapid  as  the  vibrio  proteus  of  Finkler  and 
Prior. 

On  Gelatine  Plates  small  punctiform  colonies  develop,  which  on  the 
second  day  are  about  the  size  of  a  pinhead  and  have  a  yellowish  colour ; 
examined  under  a  low  power  they  appear  coarsely  granular,  yellowish- 
green  coloured  in  the  centre  and  paler  towards  the  margins ;  funnel- 
shaped  cavities  are  formed  when  liquefaction  commences, 

In  Gelatine  Stab  Cultures  liquefaction  takes  place  along  the  inoculation 
track,  the  vibrios  sinking  to  the  bottom  of  the  medium  in  a  mass,  while 
a  thin  yellowish  layer  forms  upon  the  surface  ;  complete  liquefaction  takes 
place  in  about  two  weeks. 

On  Agar  a  yellowish-white  coating  is  formed  on  the  surface. 

Blood  Serum  is  quickly  liquefied. 

In  Bouillon  or  Peptone  Solution  the  nitroso-indol  reaction  is  awanting. 

Pathogenesis. — Fifteen  guinea-pigs  were  treated  with  soda  and 
tincture  of  opium,  the  same  as  Koch's  method  with  Asiatic  cholera,  and 
the  vibrio  introduced  into  the  intestines,  but  only  three  of  the  infected 
animals  succumbed. 


160  SPECIAL  BACTERIOLOGY 

MILLER'S   SPIRILLUM. 

This  spirillum  was  obtained  by  Miller  from  a  carious  tooth. 
Morphologically  it  is  indistinguishable  from  the  spirillum  of  Finkler 
and  Prior. 

Motility. — Non-motile. 

Staining  Reactions. — Same  as  the  vibrio  proteus. 

Biological  Characters. — On  Gelatine  Plates  small  transparent  pits 
of  liquefaction  appear  in  twenty-four  hours ;  in  the  centre  of  the 
colony  a  minute  white  speck  can  be  seen.  Examined  under  a  low 
power,  the  larger  colonies  are  granular  and  regularly  round,  and  usually 
surrounded  by  a  peripheral  zone  somewhat  darker  than  the  central 
portion  of  the  colony.  On  close  examination  the  circumference  can  be 
observed  fringed  with  short  cilia-like  growths  usually  twisted  in  all 
directions.  The  deeper  colonies  are  round,  sharply  circumscribed,  of  a 
pale  yellowish  or  greenish-yellow  colour,  and  marked  by  delicate 
irregular  lines  or  ridges.  In  forty-eight  hours  the  plate,  containing 
many  colonies,  is  liquefied. 

In  Stab  Cultures  the  gelatine  is  liquefied  very  quickly  along  the 
inoculation  track. 

On  Agar  the  growth  exhibits  nothing  characteristic. 

On  Potato,  like  the  vibrio  of  cholera,  at  37°  C.  it  forms  a  dry  white 
patch  on  the  surface,  often  only  visible  when  the  tube  is  held  to  the 
light  in  a  special  way. 

In  Bouillon  it  forms  no  pellicle. 

Solid  Blood  Serum  and  Egg  Albumen  are  liquefied. 

Glucose  is  not  fermented.     Indol  is  not  produced. 

Milk  containing  blue  litmus  tincture  is  almost  completely  decolorized 
in  from  three  to  four  days  at  37°  C.,  with  coincident  coagulation  of  the 
casein  and  the  formation  of  a  layer  of  whey  about  it. 

Pathogenesis. — Out  of  twenty-one  animals  previously  treated  by 
Koch's  method  with  soda  and  tincture  of  opium  before  infection,  only 
four  died. 

(The  other  vibrios  are  to  be  found  in  the  section  on  Water  Bacteria.) 

SPIRILLUM  OF  RELAPSING  FEVER. 

(Spirochaete  Obermeieri). 

Found  by  Von  Obermeier  in  the  blood  of  a  febris  recurrens 
patient. 

Microscopical  Appearances. — Long,  wavy,  flexible  threads,  with 


SPIRILLUM  OF  RELAPSING  FEVER  l6l 

ten  to  twenty  '  throws  '  in  its  length.     The  length  varies  from  1 6  to  40  /*, 
and  the  diameter  about  J  to  \  of  the  comma  bacillus. 

Motility. — Strongly  motile,  exhibiting  undulations  which  pass  along 
the  threads  like  a  wave. 

Staining  Reactions. — Somewhat  easily  stained  with  fuchsin, 
alkaline,  methylene  blue,  and  Bismarck  brown.  Some  spirilla,  owing 
to  their  fineness,  are  only  visible  with  a  high  power  and  strong  illumina- 
tion, while,  when  present  in  masses  in  the  blood,  they  are  easily  seen  in 
both  unstained  and  stained  preparations. 

Biological  Characters. — Outside  the  body,  in  blood  serum  and  \ 
per  cent,  chloride  of  sodium  solution,  they  retain  their  motility  for  a 
long  time.  They  have,  however,  never  been  cultivated  on  artificial 
media. 

Pathogenesis. — Monkeys  are  susceptible  when  inoculated  with 
human  blood  containing  the  spirillum,  a  typical  fever  being  produced 
during  the  height  of  the  same.  Great  numbers  of  the  spirilla  were 
present  in  the  blood,  but  were  not  either  before  or  after.  They  were 
also  found  in  the  organs  of  animals  killed  during  the  height  of  the 
fever. 

The  disease  could  be  conveyed  from  one  monkey  to  another,  but 
only  with  blood  containing  spirilla.  The  typical  fever  was  again  pro- 
duced in  a  monkey  which  received  a  second  injection  in  a  few  days  or 
weeks  after  recovery  from  the  first  injection  (Koch  and  Carter). 


VIBRIO   RUGULA. 

Found  in  faeces,  water,  and  deposit  on  the  teeth. 

Microscopical  Appearances.  —  Slightly  bent,  finely  granular, 
motile  rods,  with  flagella  (see  Photomicrograph,  Fig.  57)  situated  at  the 
end  of  the  organism  in  bundles  (Lophotricha). 

Biological  Characters. — Optimum  temperature  about  37°  C. 

On  Gelatine  Plates  it  forms  irregular  white  colonies,  the  surface 
colonies  consisting  of  delicate  tufts. 

On  Agar  the  growth  on  the  surface  of  the  medium  does  not  possess 
great  vitality,  the  development  in  the  condensation  water  being  much 
stronger. 

On  Blood  Serum,  which  is  not  liquefied,  the  growth  is  luxuriant. 

Sterile  Milk  is  not  altered. 

Indol  formation  has  not  been  observed. 

It  is  non-pathogenic. 

L 


162  SPECIAL  BACTERIOLOGY 


SPIRILLUM  UNDULA. 

Found  in  putrid  fluids,  especially  in  infusions  of  straw.  Thick, 
strongly-motile  spirilla,  the  flagella  arranged  in  a  bundle  at  one 
pole  (Lophotricha).  (See  Photomicrograph,  Fig.  58.) 

Biological  Characters. — The  optimum  temperature  appears  to  be 
between  22°  and  27°  C. 

On  Gelatine  Plates  it  forms  sharply  defined,  granular,  greenish-yellow 
colonies,  while  the  medium  appears  to  be  slightly  liquefied. 

In  Gelatine  Slab  Cultures,  on  the  upper  portion  of  the  puncture,  a 
veil-like  clouding  of  the  medium  occurs,  while  round  the  opening  of  the 
puncture  a  whitish  growth  with  ragged  tufted  edges  develops. 

On  Agar  Media  the  water  of  condensation  is  clouded,  but  no  film  is 
formed.  Kutscher  distinguishes  two  forms — Spirillum  undula  majus, 
and  Spirillum  undula  minus. 


BACILLUS  DIPHTHERIA. 

This  bacillus  was  first  observed  by  Klebs  (1883)  in  diphtheritic 
false  membranes.  It  was  cultivated  in  pure  cultures,  and  its  patho- 
genic properties  demonstrated  by  Loffler  in  1884. 

Microscopical  Appearances. — Somewhat  plump  rods  of  variable 
sizes,  1  to  6  /A  long,  and  0'5  to  1  /*  broad,  either  straight  or  slightly 
curved,  with  rounded  ends.  Irregular  forms  are  very  common,  and 
indeed  are  characteristic  of  this  bacillus.  In  the  same  culture  and  in 
unfavourable  media  great  differences  in  form  and  dimensions  occur ;  one 
or  both  ends  may  appear  swollen  (see  Photomicrograph,  Plate  IV.,  Fig. 
19,  in  the  centre  of  the  field),  or  the  central  portion  may  be  thicker 
than  the  extremities,  or  the  rod  may  consist  of  irregular,  spherical,  or 
ovoid  segments.  The  rods  sometimes  also  lie  in  clusters  alongside  of 
each  other  in  a  characteristic  manner,  like  a  bundle  of  faggots  or  a 
spilled  box  of  matches.  They  also  occur  in  branched  forms,  but  this 
condition  is  comparatively  rare. 

Spore  Formation  absent,  but  the  cultures  remain  alive  for  five 
months. 

Motility. — Non-motile. 

Staining  Reactions. — The  best  results  are  obtained  with  methylene 
blue,  or  a  weak  solution  of  carbol  fuchsin  ;  gentian  violet  stains  too 
intensely,  obscuring  the  structure  of  the  organism.  The  reaction  with 
the  Gram  and  Cladius  methods  is  positive.  Roux's  double  stain  (see 
Technique,  p.  41)  also  stains  the  bacilli  very  well.  Neisser  has-  recently 


Fio.  57.— N'ibrio  Rugula,  with  Plagella.     Agar  culture.    Stained 
with  Orcein  solution.     X  1000. 


FIG.  58.*— Spirillum  Undula,  with  Flagella.     X  1000. 
I  am  indebted  to  Herr  Hansel,  29  Dorothean  Strasse,  Berlin,  for  this  specimen. 


[T;  Bou-hill,  F.P.C.l\S.,  Photo.,  Edinburgh,  1898. 


BACILLUS  DIPHTHERIA  163 

described  a  double  stain  (see  Technique,  p.  23)  which  can  be  used  as  a 
means  of  differential  diagnosis.  In  sections  the  bacilli  can  be  stained  by 
Loffler's  method,  also  by  the  Gram  and  Cladius  methods. 

Biological  Characters. — Aerobic  and  non-liquefying,  growing  most 
freely  in  the  presence  of  oxygen,  but  is  also  a  facultative  anaerobe.  The 
growth  takes  place  only  between  20°  and  42°  C.  on  all  slightly  alkaline 
media ;  the  optimum  temperature  is  about  35°  C. 

On  Gelatine  Plates. — Small,  round,  white  colonies  develop,  which  under 
a  low  power  appear  yellowish-brown  in  colour,  and  granular,  with 
irregular  borders,  rarely  reaching  a  diameter  of  over  1-5  mm.;  the 
development  is  very  slow,  nothing  very  characteristic  being  observed  in 
less  than  seventy-two  hours. 

In  Gelatine  Stab  Cultures. — The  same  above  mentioned  ;  small,  white, 
round  colonies,  not  exceeding  the  above  size,  develop  along  the  inocula- 
tion track.  At  24°  C.  surface  growth  and  indications  of  a  nail-shaped 
growth  appear.  The  gelatine  is  not  liquefied. 

On  Agar. — Best  on  glycerine  agar  plates  ;  in  twenty-four  to  forty-eight 
hours  small,  greyish-white,  shining  colonies  develop,  which  macroscopic- 
ally  often  exhibit  a  stratified  appearance ;  and  under  a  low  power 
appear  granular,  with  irregular  borders. 

On  Agar  Stroke  Cultures. — In  twenty-four  hours  small,  transparent, 
slightly  elevated  colonies  appear  ;  the  further  growth  is  very  scanty,  and 
does  not  extend  far  from  the  inoculation  track. 

In  Agar  Stab  Cultures. — Colonies  develop  along  the  inoculation  track 
with  a  minimal  amount  of  surface  growth. 

In  a  mixture  of  glycerine  agar  and  human  blood  serum  the  growth  is 
much  more  pronounced  and  extensive  (see  Photograph  of  Culture 
growing  in  this  medium,  Fig.  59). 

On  Lqffler's  Serum  (for  preparation  of  same  see  Technique,  p.  56). — In 
twenty-four  hours  somewhat  large,  whitish  opaque  colonies  of  firm 
consistence  develop,  which  only  increase  slightly  in  size  during  the 
next  few  days.  This  medium  is  the  best  for  diphtheria  bacilli,  and  is 
always  used  for  differential  diagnosis. 

Bouillon. — In  this  medium  the  bacillus  grows  in  fine  clumps,  which 
fall  to  the  bottom  of  the  tube,  or  are  deposited  on  the  sides  without 
causing  any  clouding  of  the  medium.  The  bouillon  may  appear  diffusely 
clouded  to  the  naked  eye,  but  when  examined  microscopically  in  a 
hanging-drop  the  clumpy  arrangement  is  easily  observed.  In  bouillon 
kept  at  35°  C.  for  some  time  a  whitish  film  often  forms  over  part  of  the 
surface.  The  reaction  of  the  bouillon  is  subject  to  changes — frequently 
at  first  it  is  acid,  and  subsequently  again  alkaline.  These  changes  can  be 
observed  in  the  medium  when  a  little  rosolic  acid  is  added.  These 
reactions  are  attributed  to  the  primary  fermentation  of  muscle  sugar 
often  present  in  the  bouillon. 


164  SPECIAL  BACTERIOLOGY 

On  Potato,  which  is  rendered  alkaline,  a  delicate  coating  forms. 
Milk  is  a  favourable  medium,  and  is  not  coagulated.  Owing  to  the 
fact  that  diphtheria  epidemics  appear  sometimes  to  be  spread  by  the 
milk  supply,  Schotellius  instituted  the  following  experiments  : — 1  c.c. 
of  a  bouillon  culture  of  the  Bacillus  diphtheriae  was  added  respectively  to 
20  c.c.  bouillon,  20  c.c.  fresh  milk,  and  20  c.c.  boiled  milk.  The 
fresh  milk  was  obtained  direct  from  the  cow's  udder,  which  was 
previously  cleansed.  According  to  the  calculation  of  this  author,  1  c.c. 
of  each  of  the  three  culture  fluids  yielded  the  following  number  of 
colonies,  as  estimated  by  plate  culture  methods  after  six  hours'  cultivation 
at  ordinary  temperature  : — 

Fresh  milk          =   21,280-000  Diphtheria  colonies. 

Sterilized  milk    =      2,280-000  „  „ 

Bouillon  =      7,600-000 

After  longer  incubation  at  37°  C. : — 

Fresh  milk          =    50,160-000  Diphtheria  colonies. 

Sterilized  milk    =      6,080-000  „  „ 

Bouillon  =    18,240-000 

In  cooked  and  raw  eggs  the  diphtheria  bacillus  develops  in  both  the 
white  and  the  yelk  very  well,  also  on  solid  egg  albumen,  when  it  some- 
times exhibits  branched  forms. 

Vitality. — Corrosive  sublimate  1  to  1000  kills  cultures  in  thick  layers 
within  twenty  seconds  ;  5  per  cent,  solution  of  permanganate  of  potash, 
5  per  cent,  solution  of  carbolic  acid,  and  3  per  cent,  carbolic  acid  in  30 
per  cent,  alcohol,  in  the  same  space  of  time.  The  pure  juice  of  a  lemon 
kills  the  bacilli  very  quickly.  They  are  destroyed  when  heated  at  60°  C. 
for  ten  minutes. 

In  thick  layers  they  resist  drying  for  some  months  ;  when  in  a  dry 
state  they  die  very  quickly.  They  stand  cold  well,  but  in  the  ice-chest 
they  rapidly  lose  their  power  of  producing  a  toxine.  According  to 
Loffler  the  bacilli  remain  alive  in  gelatine  cultures  for  331  days.  In  a 
box  of  wooden  bricks  with  which  a  child  suffering  from  diphtheria 
had  been  playing  itself,  Abel  found  Loffler's  bacillus  six  months  after. 
It  has  also  been  found  in  soiled  linen,  hair,  drinking  glasses,  etc. 
Diphtheritic  membranes  dried  and  kept  in  the  dark  are,  months  later, 
capable  of  producing  cultures. 

Pathogenesis. — Under  natural  conditions  diphtheria  does  not  occur 
in  animals ;  the  so-called  spontaneous  chicken  and  pigeon  diphtheria  are 
etiologically  different  diseases.  By  inoculation  into  the  trachea  in  cats 
and  rabbits  you  get  true  diphtheritic  symptoms — general  toxaemia  and 
death  from  absorption  of  the  toxines  formed  at  the  seat  of  disease. 
Guinea-pigs  inoculated  subcutaneously  with  O'l  to  0'5  c.c.  of  a  bouillon 
culture  die  in  from  four  to  five  days,  showing  the  following  post-mortem 
appearances : — Extensive  oedema,  hyperaemia,  and  ecchymosis  at  seat  of 


BACILLUS  DIPHTHERIA  165 

inoculation,  lymphatic  glands  congested,  exudation  into  the  pleurae, 
peritoneum,  and  pericardium.  Suprarenal  capsules  are  enlarged  and 
show  hsemorrhagic  infiltration  ;  spleen  is  sometimes  enlarged  ;  also  fatty 
degeneration  of  liver  and  kidneys.  Rabbits  are  not  so  susceptible,  and 
generally  recover  after  a  small  injection.  When  death  does  not  follow 
inoculation  rapidly,  the  visceral  changes  are  less  marked,  and  we  often  get 
nervous  symptoms  with  paralysis  progressing  from  behind  forward. 
Among  common  animals  rats  and  mice  alone  are  immune,  but  MM. 
Borrel  and  Roux  found  that  though  rats  were  not  affected  by  inoculation, 
yet  if  they  injected  the  toxine  into  the  brain  the  animal  died  from 
diphtheritic  paralysis. 

Differential  Diagnosis. — In  diphtheria  we  are  specially  liable  to 
get  cases  of  mixed  infection  and  pseudo-diphtheria.  In  the  former  we 
may  have  streptococci,  staphylococci,  pneumococci,  and  B.  coli  communis. 

Pseudo-diphtheria  is  due  to  bacilli  which  resemble  very  closely  in 
their  morphology  the  true  Loffler's  bacillus,  but  are  distinguished  as 
follows : — They  are  non-pathogenic  for  experimental  animals,  and  when 
grown  in  alkaline  bouillon  do  not  change  the  reaction  of  the  medium. 
The  genuine  bacillus  changes  slightly  alkaline  bouillon  to  acid,  which 
later  (after  some  months)  again  becomes  alkaline. 

Immunity. — Fraenkel  was  the  first  to  immunize  guinea-pigs  against 
diphtheria  by  infecting  them  with  a  toxin  modified  by  exposure  to  a  tem- 
perature of  70°  C. ;  but  to  Behring  belongs  the  credit  of  the  fundamental 
discovery  that  the  blood  of  an  animal  immunised  for  a  certain  infectious 
disease  may  be  employed  for  protective  inoculation,  and  even  in  larger 
quantity  exercise  a  curative  influence  after  infection  has  occurred.  This 
is  one  of  the  greatest  discoveries  of  recent  years  in  scientific  medicine, 
even  if  the  practical  results  attained  in  human  infectious  diseases  do  not 
justify  all  the  expectations  that  were  entertained  regarding  it.  The 
above  discovery  has  been  specially  applied  to  prevent  and  mitigate  the 
ravages  of  diphtheria.  A  disease  that  can  attack  the  same  child  more 
than  once,  therefore,  does  not  belong  to  the  class  of  diseases  producing 
a  permanent  immunity  after  recovery.  It  is,  however,  well  known  that 
after  recovery  from  diphtheria  a  certain  temporary  immunity  is  conferred, 
as  the  blood  serum  of  children  during  convalescence  has  been  found  to 
possess  immunizing  properties. 

The  diphtheria  toxin  used  for  immunizing  animals  in  the  prepara- 
tion of  the  antitoxin  is  obtained  by  cultivating  the  virulent  diphtheria 
bacillus  in  bouillon  exposed  to  the  air,  or  by  making  cultivations  in  a 
current  of  moist  air  in  2  per  cent,  peptone  alkaline  bouillon  placed  in 
flat-bottomed  Fernbach  flasks,  and  sterilized  previous  to  inoculation. 
In  three  weeks,  or  longer,  the  culture  is  rich  enough  in  toxins  to  be 
employed.  The  culture  is  next  filtered  through  a  Chamberland  filter, 
and  the  clear  filtrate  preserved  in  vessels  well  filled,  and  protected  from 


166  SPECIAL  BACTERIOLOGY 

the  light  and  kept  at  ordinary  temperature.  One-tenth  c.c.  of  a  toxin 
so  prepared  is  usually  fatal  in  forty-eight  hours  to  a  guinea-pig  weighing 
500  grammes.  It  loses  its  activity  after  a  time,  though  very  slowly  if 
kept  in  the  manner  above  mentioned. 

There  is  considerable  variance  in  the  degrees  of  virulence  of  different 
cultures  of  diphtheria  bacilli.  This  varying  virulence  tends  to  explain 
the  protean  nature  of  the  disease  and  the  differing  character  of  the 
various  epidemics.  It  also  helps  to  explain  the  occurrence  of  diphtheria 
bacilli  in  '  rhinitis  fibrinosa  '  and  other  benign  affections. 

Experiment  animals  are  easily  immunized.  Behring,  and  later  Roux, 
immunized  horses  with  diphtheria  toxin  which  had  its  poisonous  proper- 
ties weakened  by  the  addition  of  a  solution  of  trichloride  of  iodine,  or 
iodide  of  potassium. 

A  serum  possessing  very  high  immunizing  properties  is  obtained  from 
the  horse  by  introducing  into  that  animal  large  quantities  of  diphtheria 
toxin  as  follows : — 


ROUX'S   METHOD    OF   IMMUNIZING   A   HORSE. 

Subject. — Seven-year  old  horse,  weighing  about  400  kilogrammes. 
The  toxin  used  was  very  active ;  ^  c.c.  killed  in  forty-eight  hours  a 
guinea-pig  weighing  500  grammes.  The  point  of  injection  was  under 
the  skin  of  the  neck  or  behind  the  shoulders. 


Days  of  Injection.       Injection  of    ^^ttj^£dk  Reaction. 

1  %    c.c.                 1 — 10  No  reaction. 

2  \     ,,                  1—10  do. 
4,  6,  8                    \      „                   1—10  do. 
13,  14                    1      „                   1—10  do. 

17                          i     »»             Pure  toxin.  Slight  oedema,  no  fever. 

22  1      „                    do.  do. 

23  2      „                     do.  do. 
25                           3      „                     do.  do. 
28                           5      „                     do.  do. 
30,  32,  36              5      „                     do.  do. 
39,  41                   10      „                     do.  do. 

43  46   48   50      30                            do  Well-marked  oedema,  disappearing  in 

43,  46,  48, 5C               „  twenty-four  hours. 

53                         60      „                     do.  do. 

57,  63,  65,  67      60      „                     do.  do. 

72                         90     ,,                     do.  do. 

80                       250      „                     do.  do. 

In  eight  to  ten  days  after  the  last  injection  5  to  6  litres  of  blood 

are  taken  from  the  jugular  vein  with  a  sterilized  trocar,  and  placed  in 
the  ice-chest,  when  a  clear  serum  forms. 

Behring  preserves  the  serum  thus  obtained  by  adding  0*5  per  cent, 
of  carbolic  acid.  Schering's  serum  is  preserved  with  0*4  per  cent,  of 


ROUX'S  METHOD  OF  IMMUNIZING  A  HORSE          167 

trikresol  which  is  considered  to  possess  about  twice  the  antiseptic  power  of 
carbolic  acid,  and  is  only  half  as  poisonous,  while  in  the  Pasteur  Institute 
they  use  a  piece  of  camphor.  Serum  desiccated  in  vacua  is  convenient 
to  send  to  a  distance,  and  reacquires  its  preventive  properties  when 
dissolved  again  in  eight  or  ten  times  its  weight  of  pure  water. 

Instructions  for  the  use  of  Schering's  Diphtheria  Anti- 
toxin.— A  hypodermic  syringe  is  used,  holding  10  to  12  c.c.,  which, 
before  use,  must  be  thoroughly  asepticized  with  alcohol  and  a  1  per 
cent,  solution  of  trikresol. 

The  injection  is  made  deep  into  the  subcutaneous  connective  tissue  of 
the  skin,  the  back,  between  the  shoulder  blades,  or  the  thighs,  being  the 
points  of  election.  Before  the  injection  is  made,  the  skin  is  scrubbed 
with  soap  and  water,  and  then  with  ether.  After  the  injection  the 
puncture  may  be  immediately  sealed  with  rubber  plaster,  or  cotton  and 
iodoform  collodion,  to  prevent  any  loss  of  fluid  through  the  dermal 
puncture. 

For  the  immunization  of  children  and  adults  in  families  where 
diphtheria  has  occurred,  isolation  being  unnecessary,  and  also  for 
general  use  among  those  who  have  been  exposed  in  epidemics,  the  dose 
is  as  follows  : — 

Up  to  two  years  of  age,  0'5  c.c.     (8    minims). 

From  two  to  ten  years  of  age,  Ic.c.    (16     do.     ). 
Over  ten  years,  and  for  adults,  2  c.c.   (32     do.     ). 

If  in  four  to  six  weeks  the  epidemic  has  not  ended,  it  is  well  to 
repeat  the  injection. 

For  the  Cure  of  Diphtheria  extensive  practice  has  shown  that  a  single 
injection  of  5  c.c.  (80  minims)  of  the  solution  at  present  supplied  is 
certainly  curative  if  used  at  the  beginning  of  the  disease.  In  severer  cases 
that  have  lasted  some  time,  two  injections  must  be  given  in  the  course 
of  twelve  to  twenty-four  hours,  of  5'20  c.c.  (80  minims  to  5j  fluid 
drachms),  according  to  the  body-weight.  In  some  cases  still  larger 
doses  have  been  successfully  employed.  The  further  repetition  of  the 
injection  depends  upon  the  following  factors  : — 

a.  The  course  and  increase  of  the   exudation,   and   the   condition  of 

the  glands. 

b.  The  course  of  the  temperature. 

c.  The  condition  of  the  kidneys. 

d.  The  condition  of  the  pulse  and  the  heart  sounds. 


BACILLUS   DIPHTHERIA   COLUMBARUM. 

Obtained  by  Loffler  in  1884  from  the  false  membranes  in   the 
mouths  of  pigeons.     Chickens  are  also  affected. 


168  SPECIAL  BACTERIOLOGY 

Symptoms. — In  pigeons,  reddened  patches  on  mucous  membrane  of 
mouth  and  fauces,  which  are  covered  later  with  a  layer  of  thick  yellow 
fibrinous  exudation,  the  back  part  of  tongue,  fauces,  and  corners  of 
the  mouth  being  specially  affected.  In  chickens  the  tongue,  gums,  nares, 
larynx,  and  conjunctival  mucous  membranes  are  the  parts  affected 
(see  Photo.,  Fig.  60,  of  two  Plymouth  Rock  chickens  suffering  from 
this  disease).  The  disease  is  very  fatal  to  young  fowls,  the  choice 
varieties  being  most  susceptible. 

Microscopical  Appearances. — Short  bacilli  with  rounded  ends 
usually  grouped  together.  They  are  longer  and  narrower  than  the 
bacillus  of  chicken  cholera.  Sections  of  liver  show  them  in  irregular 
masses  in  the  interior  of  the  vessels. 

Motility. — Non-motile. 
Spore  Formation  absent. 

Staining  Reactions. — Stain  with  ordinary  aniline  dyes.  Gram's 
reaction  negative. 

Biological  Characters. — Aerobic  non-liquefying  bacillus. 

On  Gelatine  Plates  greyish  white  colonies,  which  under  a  low  power 
resemble  the  typhoid  bacillus. 

In  Gelatine  Stab  Cultures,  grows  like  a  nail,  with  a  whitish  head. 

On  Egg,  Agar-Agar,  Potato  and  Blood  Serum  it  forms  a  greyish 
covering. 

Bouillon  is  clouded,  but  there  is  no  indol  reaction. 

Pathogenic  for  rabbits,  mice,  small  birds,  pigeons,  and  chickens. 
Rats  are  only  slightly  affected. 

There  are  white  masses  of  necrosed  liver  tissue  in  the  livers  of  mice 
in  whom  the  disease  has  been  inoculated  containing  large  numbers  of 
bacilli  in  the  interior  of  the  vessels.  This  characteristic  is  considered 
by  Loffler  to  be  the  best  method  of  identifying  the  bacillus. 


DIPHTHERIA  VITULORUM. 

Obtained  by  Loffler  in  1884  in  false  membranes  from  the  mouths 
of  calves  suffering  from  an  infectious  form  of  diphtheria. 

Symptoms. — Yellow  patches  on  mucous  membrane  of  cheeks,  gums, 
tongue,  sometimes  of  larynx  and  nares,  yellow  discharge  from  the  nose, 
excessive  salivation,  occasional  coughing,  and  diarrhoea.  The  animal 
may  die  in  four  or  five  days,  or  may  survive  for  several  weeks.  Diph- 
theritic patches  like  those  in  mouth,  etc.,  occur  in  the  large  intestine, 
and  sometimes  abscesses  are  found  in  the  lungs. 


FIG.  59.  B.  Diphtheria-. 
Human  blood  serum  a^ar 
culture. 


Fm.  61.—  B.  Typhi  Abdominalis.    Gela- 
tine stab  culture. 


Fio.  60.— Avian  Diphtheria.    Chickens  in  the  last  stages  of  the  disease. 

\T.  Jtowhill,  F.R,C.V.S.,  Photo.,  Edinburgh,  1898, 


DIPHTHERIA  VITULORUM  169 

Microscopical  Appearances. — Bacilli  five  or  six  times  as  long  as 
broad,  usually  in  filaments. 

Biological  Characteristics. — This  bacillus  does  not  grow  in 
nutrient  gelatine  blood  serum  from  sheep  and  other  usual  media,  but 
in  the  blood  serum  of  calves  pieces  of  the  affected  tissue  gave  a  whitish 
growth  of  the  bacillus,  which,  however,  did  not  grow  when  transferred 
afresh  to  serum. 

Pathogenesis. — Fatal  for  mice  in  from  seven  to  thirty  days  when 
inoculated  subcutaneously,  the  autopsy  revealing  extensive  infiltration 
of  abdominal  walls,  which  often  spreads  into  the  peritoneal  cavity, 
enveloping  the  viscera  in  a  yellowish  exudation.  The  bacilli  are  formed 
in  this  exudation,  and  mice  inoculated  with  some  of  the  fluid  die 
similarly.  Non-pathogenic  for  rabbits  and  guinea-pigs. 


BACILLUS  TYPHI  ABDOMINALIS. 

(Bacillus  of  Typhoid  Fever.) 

This  organism  was  first  observed  by  Eberth  in  the  internal  organs 
of  typhoid  cadavera.  Koch  also  saw  the  bacillus  about  the  same 
time,  and  photographed  it.  It  was  first  obtained  in  pure  cultures  by 
Gaffky,  and  has  also  been  found  during  life  in  the  blood,  urine,  and 
faeces  of  typhoid  patients. 

Microscopical  Appearances.  —  Short,  plump  rods  with  rounded 
ends,  1  to  3  ft  long,  and  0'5  to  0'9  /*  broad,  which  in  sections  of  tissue 
are  usually  found  singly,  but  in  cultures  often  found  in  long  threads. 
(See  Photomicrograph,  Fig.  62.)  In  agar  cultures  at  37°  C.,  in  the 
bodies  of  animals,  and  in  human  tissues,  the  rods  are  more  plump,  and 
smaller  in  all  directions  than  on  gelatine  and  potato  media,  and  the  long 
threads  occur  more  frequently  at  lower  temperatures. 

Motility. — Actively  motile,  each  bacillus  possessing  eight  to  eighteen 
flagella  situated  along  the  sides  and  ends  (Peritricha).  (See  Photo- 
micrograph, Plate  II.,  Fig.  7,  stained  by  the  author's  orcein  method  ;  also 
the  same  preparation  x  1500  diameters,  Plate  II.,  Fig.  8.) 

Spore  Formation  does  not  exist.  The  so-called  spores  of  Gaffky 
are,  according  to  later  investigations,  involution  forms. 

Staining  Reactions. — With  the  ordinary  aniline  dyes  the  rods  do 
not  stain  so  readily  as  most  other  organisms.  Watery  solutions  of  the 
dyes  and  a  weak  solution  of  carbol  fuchsin  give  the  best  results  when 
slightly  heated  during  the  staining  process.  They  do  not  stain  by  the 
Gram  method.  Small  vacuoles  are  sometimes  present  in  the  rods,  due 
to  retraction  of  the  protoplasm  from  the  cell  envelope  of  the  bacilli. 


170  SPECIAL  BACTERIOLOGY 

The  bacilli  are  easily  detected  in  the  tissues,  especially  in  pieces  of  the 
liver  or  spleen,  where  they  can  be  observed  massed  together  in  charac- 
teristic clumps  when  stained  with  carbol  fuchsin  at  the  ordinary  room 
temperature,  or  at  40°  to  45°  C.,  washed  in  absolute  alcohol,  cleaned  in 
xylol,  and  mounted  in  xylol  balsam.  Alkaline  methylene  blue  can  also 
be  used.  The  characteristic  clumps  in  the  tissues  consist  of  small  ovals 
or  rods  closely  packed  together,  individual  bacilli  being  often  only  visible 
at  the  periphery  of  the  mass,  usually  in  the  neighbourhood  of  a  capillary. 

Biological  Characters. — It  grows  at  any  temperature  between  20° 
and  38°  C.  on  the  ordinary  nutrient  media.  The  growth  is  most 
luxurious  at  incubator  temperature,  while  at  ordinary  room  temperature 
the  development  is  very  slow.  It  also  grows  both  with  and  without 
oxygen  (facultative  anaerobe),  and  fairly  well  in  CO2  ;  and  as  a  contrast 
to  most  other  pathogenic  bacteria,  grows  luxuriantly  on  slightly  acid 
media. 

On  Gelatine  Plates  the  deep  colonies  are  small,  punctiform,  and  sharply 
circumscribed  ;  under  a  low  power  they  exhibit  a  brownish  colour.  The 
superficial  colonies  are  much  larger,  forming  a  bluish-white,  iridescent, 
fine  coating  with  irregular  borders,  denser  in  the  centre  than  at  the 
periphery,  and  under  a  low  power  exhibit  a  brownish  colour  and  wrinkled 
appearance.  The  gelatine  is  not  liquefied. 

In  Gelatine  Stab  Cultures  the  growth  is  mostly  limited  to  the  surface, 
with  limited,  thready,  granular  growths  down  the  track  of  the  needle, 
often  of  a  yellow  or  yellowish-brown  colour  (see  Photograph,  Fig.  6l). 

On  Gelatine  Stroke  Cultures  a  fine,  iridescent,  bluish  growth  extends 
from  the  centre,  and  soon  covers  the  whole  surface  of  the  gelatine. 
The  gelatine  is  not  liquefied. 

On  Agar  and  Blood  Serum  Media  an  extrusive,  thick  coating  develops 
which  presents  no  typical  characteristics. 

On  Potato. — The  growth  upon  the  surface  of  a  cut  potato  appears  as 
if  nothing  had  developed ;  but  if  examined,  it  will  be  found  that  the 
whole  surface  of  the  potato  is  covered  with  tufts,  which,  when  examined 
microscopically,  will  be  found  to  consist  of  numerous  motile  rods.  This 
peculiarity  of  growth  occurs,  as  far  as  is  known  at  present,  exclusively  in 
this  bacillus.  Sometimes,  though  rarely,  the  growth  is  visible,  for  there 
are  some  kinds  of  potatoes  upon  which  the  bacilli  developed  a  raised 
circumscribed  tuft  of  a  yellowish  or  brownish  colour.  These  potatoes 
possess  either  a  neutral  or  alkaline  reaction,  while  the  typical  growth  is 
confined  to  those  exhibiting  an  acid  reaction. 

In  Milk  Media  an  acid  reaction  results  from  the  growth  without 
causing  coagulation,  whereas  the  coli  commune  causes  an  acid  reaction 
and  coagulation  in  twenty-four  to  forty-eight  hours  at  37°  C. 

Bouillon  is  clouded,  with  a  quantity  of  sediment  and  slightly  acid 
reaction. 


BACILLUS    TYPHI  ABDOMINALIS  171 

In  Grape,  Milk,  and  Cane  Sugar  Media  no  fermentation  takes  place, 
and  according  to  Hellstrom,  in  media  containing  4  per  cent,  milk  sugar 
and  1  to  4  per  cent,  peptone,  inoculated  with  the  typhus  bacillus,  and 
kept  at  37°  C.  for  twenty-four  hours,  the  colonies  developing  are  much 
smaller  than  those  of  the  coli  commune  under  similar  conditions. 

On  Halzs  Potato  Gelatine  (for  preparation  of  same,  see  Technique, 
§  79)  the  growth  of  this  bacillus  and  the  coli  commune  is  more 
pronounced  than  that  of  other  ordinary  bacteria.  To  this  medium 
Eisner  added  1  per  cent,  iodide  of  potassium  (see  Technique,  §  79). 
On  this  medium  the  coli  commune  grows  more  energetically  than  the 
typhoid  bacillus,  exhibiting  dark  brown  colonies  in  forty-eight  hours, 
whereas  the  colonies  of  the  typhoid  bacillus  appear  as  clear,  watery  drops. 
This  growth  is  however  not  absolutely  constant,  further  identification 
with  other  culture  methods  being  necessary. 

Vitality. — In  sterilized  water  the  typhoid  bacilli  live  as  long  as 
three  months,  and  increase  in  numbers  at  first ;  in  ordinary  water  they 
are  destroyed  by  the  concurrence  of  the  ordinary  water  bacteria  in  about 
fourteen  days  ;  in  running  water  this  takes  place  more  quickly.  Under 
favourable  circumstances,  protected  from  light  and  drying,  they  live  a 
long  time.  In  faeces  they  appear  to  live  three  months  or  more,  depend- 
ing upon  the  number  of  putrefactive  organisms  present.  They  can 
withstand  cold  very  well ;  freezing  and  thawing  two  or  three  times  does 
them  no  harm.  They  are  not  so  resistant  to  heat,  being  destroyed  with 
certainty  in  ten  minutes  at  60°  C.,  and  in  a  shorter  time  at  higher 
temperatures. 

Specific  Reactions. — 1.  The  indol  reaction  does  not  exist  except  by 
Peckham's  method  of  repeated  transplantation  at  short  intervals  into 
either  Dunham's  peptone  solution,  or  freshly  prepared  alkali  tryptone 
solution.  Chantemasse  has  also  seen  a  red  colour  produced  in  old 
cultures  with  the  addition  of  the  indol  reagents. 

2.  Produces  no  formation  of  gas  in  the  culture  media.     This  test  is 
made  with  grape  sugar  bouillon  and  a  fermentation  tube  at  37°  C. 

3.  On  lactose-litmus-agar,   pale  blue  colonies  develop  with  no  red- 
dening  of  the  surrounding  medium ;  but  if  glucose  is  used  instead  of 
lactose,  both  the  colonies  and  the  surrounding  medium  become  red. 

4.  Gruber's  Reaction. — When  immune  serum  is  added  to  a  bouillon 
culture  of  the  suspected  bacillus  in  the  proportion  of  one  to  forty,  in 
twenty-four  hours,  if  the  organism  is  the  typhoid  bacillus,  appearances  of 
agglutination  are  evident ;  the  bacteria  form  granular  masses  at  the 
bottom  of  the  test-tube,  while  the  upper  portions  of  the  medium  remain 
clear.     These  phenomena  can  be  further  examined  microscopically,  and 
the  agglutination  and  loss  of  motility  of  the  organisms  confirmed. 

Widal's  Reaction. — The  following  is  the  method  of  making  the  test : — 
A  drop  of  blood  is  taken  from  the  ear  or  finger  of  the  suspected  typhoid 


172  SPECIAL  BACTERIOLOGY 

in  a  U-shaped  tube  and  centrifugalised,  the  tube  is  broken  off  at  the 
junction  of  the  serum  and  corpuscles,  and  the  drop  of  serum  blown  on 
to  a  glass  slide.  The  necessary  quantity  is  sucked  up  to  the  first  mark 
on  a  special  straight  capillary  tube,  with  another  mark  corresponding 
to  sixteen  times  the  volume  at  the  first  mark.  Bouillon  is  then  sucked 
up  with  the  serum  until  the  second  mark  is  reached,  and  the  whole 
blown  on  to  a  glass  slide,  mixed,  and  again  sucked  up ;  and  the  process 
repeated  two  or  three  times  to  ensure  thorough  mixing.  The  emulsion 
of  typhoid  fever  bacilli  is  prepared  by  taking  a  small  platinum  loop  full 
of  a  culture  not  more  than  twenty-four  hours  old,  grown  on  rather  dry 
agar,  and  carefully  rubbing  it  up  against  the  side  of  the  glass  tube  con- 
taining 1  c.c.  of  bouillon  with  1  drop  of  the  bouillon,  and  subsequently 
mixing  it  with  the  whole  quantity.  A  control  preparation  is  examined 
microscopically  to  make  sure  there  are  not  any,  or  very  few  pre-existent 
clumps  in  the  emulsion.  A  small  drop  of  the  diluted  serum  is  then  placed  on 
a  cover-glass,  and  a  drop,  as  near  the  same  size  as  possible,  from  the  typhoid 
emulsion  mixed  with  it,  and  a  hanging-drop  specimen  prepared  in  the 
usual  manner.  Microscopically  examined,  the  bacilli  will  be  observed 
to  gradually  form  groups  of  three  or  four,  which,  with  the  addition  of 
other  bacilli,  constantly  increase  in  size,  until  the  majority  are  in  c  clumps  ' 
with  impaired  or  lost  motility.  If  the  reaction  is  marked  within  thirty 
minutes  the  case  is  one  of  enteric  fever,  but  without  great  experience 
it  is  impossible  to  say  that  the  absence  of  this  reaction  negatives  such  a 
diagnosis.  In  negative  results  more  than  one  examination  should  be 
made,  for  it  occasionally,  although  rarely,  occurs  that  probably  from 
experimental  errors,  such  as  varying  quality  of  bouillon,  etc.,  that  the 
reaction  is  seen  on  one  day  and  not  on  another. 

Wyatt  Johnston  has  published  results  got  by  using  a  watery  solution 
of  long-dried  blood  serum  from  typhoid  patients.  The  crust  of  blood 
is  covered  with  a  drop  of  water,  and  on  standing  for  one  or  two  minutes 
a  drop  of  this  is  mixed  with  one  loopful  of  a  typhoid  culture,  a  second 
loopful  being  added  later.  The  agglutinative  action  is  seen  as  in  the 
ordinary  way  of  doing  Widal's  reaction. 

In  1897,  the  author,  whilst  working  this  test  in  California  with  Prof. 
Kerr  of  the  California  University,  found  that  quite  good  results  were  got 
by  using  filter  paper  to  absorb  the  blood,  and  then  making  a  watery 
solution  as  above.  This  method  was  much  more  convenient  for  most 
ordinary  cases  than  the  capillary  tubes  with  ceiitrifugalisation. 

The  Investigation  of  Water  for  Typhoid  Bacilli. — This  is  essential  in  all 
outbreaks  of  typhoid  fever,  for  water  has  been  repeatedly  shown  to  be 
the  vehicle  of  infection.  For  this  examination,  carbolic  acid  is  added  to 
the  water  in  the  proportion  of  0'05  to  0'25  per  cent.,  as  this  addition 
serves  to  inhibit  the  development  of  the  ordinary  liquefying  water 
bacteria,  while  the  typhoid  and  some  allied  forms  grow  in  presence  of  a 


BACILLUS  TYPHI  ABDOMINALIS  173 

small  amount  of  phenol.  Plate  cultures  are  then  instituted  with  the 
carbolized  water  after  the  method  of  Eisner. 

In  this  examination  it  is  apparent  that  to  get  positive  results  large 
quantities  of  the  water  may  have  to  be  examined,  and  this  is  best  done 
thus : — An  alkaline  sterilized  solution  made  with  peptone  and  common 
salt,  containing  1  gramme  of  each  in  a  given  volume,  is  taken.  With 
this  you  mix  100  c.c.  of  the  carbolized  water  supply,  and  place  in  sterile 
Erlenmeyer  flasks,  and  put  in  the  incubator  for  eighteen  to  twenty- 
four  hours.  Under  these  conditions  the  growth  of  any  typhoid  bacilli  is 
allowed  to  go  on  practically  without  competition,  and  the  bulk  of  the 
mixture  gives  greater  chances  of  their  appearing  in  plate  cultures. 

This  system  enables  one  to  separate  out  chiefly  typhoid  and  pseudo- 
typhoid  forms  as  well  as  coli  commune,  which  is  really  more  resistant  to 
phenol  than  is  typhoid  itself,  and  further  researches  must  be  instituted 
with  the  individual  colonies  to  get  absolute  differentiation. 

Pathogenesis. — When  some  virulent  culture  is  introduced  in 
mice,  guinea-pigs,  rabbits,  and  goats,  death  occurs  with  the  following 
symptoms : — Spasms,  falling  temperature,  and  diarrhoea.  By  sub- 
cutaneous injection  large  quantities  of  the  culture  are  necessary,  but  by 
intraperitoneal  and  intravenous  methods  a  small  quantity  is  sufficient. 
Experimental  results  in  the  lower  animals  show  that  in  most  cases 
death  occurs  without  the  appearance  of  typical  pathological  changes, 
the  fatal  result  in  most  cases  being  due  to  toxic  rather  than  to 
infective  action  of  the  virus.  Cygnaeus  introduced  typhoid  bacilli 
into  the  tissues  of  dogs,  rabbits,  and  mice,  and  produced  changes  in 
the  small  intestines,  histologically  and  macroscopically  analogous  to 
those  found  in  the  human  subject.  Abbot  obtained  only  one  positive 
result  out  of  a  large  number  of  experiments,  producing  an  ulcer  in  the 
ileum  of  a  rabbit,  macro-  and  microscopically  identical  with  that  found 
in  man.  Cultivations  were  obtained  from  the  spleen,  and  the  typical 
bacilli  demonstrated  in  characteristic  clumps  in  sections  of  the  same. 
Sanarelli  found  that  rabbits,  guinea-pigs,  and  mice  were  rendered 
susceptible  to  infection  when  first  inoculated  with  the  products  of  the 
growth  of  certain  saprophytes,  Proteus  vulgaris,  Bacillus  prodigiosus, 
and  Bacillus  coli  communis,  and  when  subsequently  fresh  cultures  of 
the  typhoid  bacillus  were  introduced  intravenously  or  into  the 
peritoneal  cavity,  death  resulted  in  twelve  to  forty-eight  hours,  with 
well-marked  pathological  changes  in  the  digestive  tract,  especially  in 
the  small  intestines.  The  infection  is  general  in  those  cases,  and  the 
bacilli  can  be  recovered  from  the  blood  and  internal  organs.  Sanarelli 
considers  that  the  toxic  condition  produced  by  the  absorption  of  the 
products  of  the  saprophytes  may  be  analogous  to  a  similar  condition 


174  SPECIAL  BACTERIOLOGY 

occurring  in  man  from  absorption  of  abnormal  products  of  fermenta- 
tion from  the  intestinal  canal.  An  auto-intoxication  whereby  the 
resistance  of  the  individual  to  infection  by  the  typhoid  bacillus,  should 
it  gain  access  to  the  alimentary  tract,  is  reduced. 

BACTERIUM    COLI    COMMUNIS. 

(Bacillus  Neapolitanus.     The  Colon  Bacillus.) 

This  bacillus  is  a  normal  inhabitant  of  the  intestines  of  man, 
cattle,  swine,  and  dogs.  It  is  also  found  associated  with  diseased 
conditions,  such  as  inflammatory  and  suppurating  processes  in  the 
peritoneal  cavity,  infectious  enteritis,  affections  of  the  liver,  puerperal 
fever,  broncho-pneumonia,  empyema,  endocarditis,  meningitis,  cystitis, 
and  pyelo-nephritis. 

Microscopical  Appearances. — Occurs  in  short  motile  rods,  mostly 
in  pairs  (see  Photomicrograph,  Fig.  64),  sometimes  in  threads,  and  vacu- 
oles  are  frequently  present  in  the  middle  of  the  rod  as  unstained  spots. 

Motility. — The  rods  possess  numerous  long  flagella  (see  Photomicro- 
graph, Plate  II.,  Fig.  11). 

Spore  Formation  is  absent. 

Staining  Reactions. — Stains  with  the  ordinary  aniline  dyes,  but 
not  by  either  the  Gram  or  Cladius  method. 

Cultivation. — In  cultures  the  organism  grows  under  both  aerobic  and 
anaerobic  conditions,  and  in  the  latter  condition  forms  gas  in  the  media. 

On  Gelatine  Plates  it  forms  iridescent  colonies  with  wavy  bent 
borders  on  the  surface,  while  the  deeper  colonies  are  round  and  of  a 
brownish  colour ;  the  gelatine  is  not  liquefied. 

In  Gelatine  Stab  Cultures  it  grows  in  the  form  of  a  nail  with  limited 
growth  along  the  track  of  the  needle  (see  Photograph  of  Culture,  Fig.  63). 

On  Agar-Agar  it  forms  a  grey  coating. 

On  Potato  the  growth  is  of  a  brownish  colour. 

In  Bouillon  it  causes  diffuse  clouding. 

In  Milk  Media  it  causes  coagulation,  which  generally  takes  place  in 
about  thirty-six  hours. 

Special  Reactions. — Indol  is  formed  by  adding  1  c.c.  of  a  2  per 
cent,  solution  of  potassium  nitrite  and  a  little  sulphuric  acid  to  10  c.c.  of 
a  bouillon  culture,  when  a  rose-red  colour  results.  It  also  produces  indol 
in  Dunham's  peptone  solution  in  forty-eight  to  seventy-two  hours.  This 
organism  also  causes  fermentation  in  milk  sugar,  grape  sugar,  and 
glycerine  media. 

On  Lactose-Litmus- A  gar,  the  colour  of  the  colonies  is  pink,  and  that  of 
the  surrounding  medium  blue-red. 

Pathogenesis. — Affects  mice,  guinea-pigs,  and  rabbits,  the  strength 


i     .. 

'.^'SS 


Fia.  (52.— B.  Typhi  Abdominalis.     Agar  culture.     Fuchsin.     X  1000. 


Fio.  M.     B.  Coli  Coinmunis.     Ayar  culture. 
Fuchsin.     X  1200. 


FIG.  63.— B.  Coli  Commnnis.     Gelatine 
stab  culture, 


[T.  Bou'hiU,  F.R.C.V.S.,  Photo.,  Edinlnirgh,  1898, 


BACTERIUM  COLI  COMMUNIS 


175 


of  the  virus  varying  according  to  the  severity  of  the  processes  from  which 
it  was  originally  isolated. 

For  the  differentiation  of  the  Bacillus  coli  communis  and  Bacillus 
typhi  abdominalis,  Piorkowski  institutes  cultures  in  bouillon,  gelatine, 
and  agar,  prepared  with  urine,  to  which  the  desired  quantity  of  peptone, 
gelatine,  and  agar  is  added  ;  sterilization,  etc.,  being  accomplished  in 
the  ordinary  manner. 

Cesaris-Demel  (Giornale  delta  R.  Accad.  di  Medicina  di  Torino,  1898, 
No.  3)  describes  a  new  method  for  the  differential  diagnosis  of  the 
typhus  bacillus  and  the  B.  coli  communis  as  follows : — 

Cultures  of  the  organisms  are  instituted  in  bouillon  prepared  from  a 
calf  s  liver.  The  sugar  present  in  the  liver  will  be  fermented  by  the 
B.  coli  communis,  and  it  will  exhibit  a  quick  luxurious  growth,  while  on 
this  medium  the  growth  of  the  typhus  bacillus  is  confined  to  very  narrow 
limits. 

DIFFERENTIAL  DIAGNOSIS   TABLE. 


BACILLUS  TYPHI  ABDOMINALIS 

BACTERIUM  COLI  COMMUNIS 

(1)  Motility 

Usually  very  pronounced  ;  large 
numbers  of  flagella,  staining 
without  much  difficulty  with 
orce'in 

As  a  rule  not  very  pronounced, 
sometimes   absent  ;     flagella 
demonstrated  with  difficulty, 
fewer  and  shorter 

(2)  Cultures 

On  gelatine  plates  the  typhoid 
colonies    develop    somewhat 
slowly,     while    on    potatoes 
they  are  as  a  rule  invisible 
(not  always) 

Does  not  coagulate  milk  with 
an  acid  reaction 

On  gelatine  the  colon  bacillus 
develops  more  rapidly  than 
the  typhoid,  and  on  potatoes 
it  grows  luxuriantly  and  is 
always  visible 

Coagulates     milk     with     acid 
reaction  in  thirty-six  to  forty- 
eight  hours  in  the  incubator 

(3)  Fermentation 

Causes    no    gas    formation    in 
media     containing     glucose, 
lactose,  or  saccharose 

Forms  gas  in  glucose,  lactose, 
and  saccharose  media 

(4)  Changes  observed 
in  agar  or  gelatine 
medi  um  containing 
lactose  and  litmus 
tincture  of  slightly 
alkaline  reaction 

The  colonies  are  of  a  pale  blue 
colour,  and  there  is  no  red- 
dening   of    the    surrounding 
medium 

The  colonies  are  pink,  and  the 
surrounding  medium  red 

(5)  Indol  test 

Does  not  as  a  rule  produce  indol 
in  solutions  of  peptone 

Produces  indol  in  solutions  in 
forty-eight    to    seventy-two 
hours  at  37-38°  C. 

(6)  Widal's  reaction 

When    a    twenty-four-hour-old 
bouillon  culture  is  placed  in 
contact  with  a  genuine  case 
of  typhoid   fever,  after  the 
fifth  day  of  the  disease  the 
characteristic     agglutination 
or  clumping  occurs 

The  reactions  toward  this  test 
are  negative 

.  (7)  Pfeiffer's  reaction 
with  typhoid  serum 

Positive 

Negative 

176  SPECIAL  BACTERIOLOGY 

Kashida  has  recently  observed  that  the  acid  produced  by  the  colon 
bacilli  in  cultures  becomes  neutralised  in  a  little  time,  an  alkaline 
reaction  soon  appearing,  which  was  so  strong  that  colonies  on  litmus 
milk  sugar  agar  plates  were  coloured  an  intense  blue,  and  when  rosolic 
acid  was  used  as  a  reagent,  the  colonies  were  coloured  red.  With  the 
Bacillus  typhi  abdominalis  the  above  characteristic  is  awanting.  Investi- 
gations of  the  condensation  water  of  agar  cultures  showed  that  the 
alkaline  reaction  of  the  colon  bacilli  was  due  to  the  formation  of  ammonia. 
This  was  very  distinct  in  cultures  on  1  \  per  cent,  meat  infusion  agar  to 
which  2  per  cent,  milk  sugar,  1  per  cent,  urea,  and  30  per  cent,  litmus 
tincture  were  added. 


BACILLUS   EQUI   INTESTINALIS. 

Found  by  Dyas  and  Keith  in  the  intestines  of  a  horse.  It  is 
distinguished  from  the  coli  communis  as  follows :  It  is  somewhat  thicker, 
does  not  grow  at  a  low  temperature,  and  in  fermentation  tubes  produces 
no  gas,  but  it  coagulates  milk  in  one  to  two  days. 

Systematic  examination  of  the  alimentary  contents  of  both  man  and 
animals  shows  that  numerous  bacteria  are  present ;  but  Nuttall  and 
Thierfelder  discovered  in  a  course  of  experiments  that  animals  could 
live  without  the  presence  of  bacteria  in  the  alimentary  canal.  The 
experiments  were  conducted  with  young  guinea-pigs,  which  were  born 
by  Ccesarean  section  to  prevent  bacteria  entering  their  economy,  nourished 
with  sterile  food,  killed  in  eight  days,  and  examined  for  bacteria,  the 
results  being  negative. 

INFLUENZA. 

Pfeiffer  discovered  the  bacillus  and  isolated  it  in  pure  cultures 
during  the  epidemic  in  1891-92.  Pfeiffer's  discovery  has  been  fully 
confirmed  by  others. 

Microscopical  Appearances. — Extraordinarily  small  bacilli  (0-2  to 
0'5  ft),  and  only  two  to  three  times  as  long  as  broad,  with  rounded  ends. 
It  very  seldom  forms  threads  in  sputum,  but  frequently  in  fresh  pure  cul- 
tures ;  in  three  to  four-day-old  cultures  involution  forms  are  already 
visible.'  Two  particularly  small  bacilli  are  frequently  observed  arranged 
close  together,  causing  them  to  be  easily  mistaken  for  diplococci.  (For 
Photomicrograph  of  the  bacilli  in  sputum,  see  Fig.  65.) 

Motility. — Non-motile. 

Spore  Formation  does  not  appear  to  exist ;  it  has  never  been  ob- 
served in  either  secretions  or  cultures,  and,  moreover,  the  bacillus  exhibits 
only  slight  resistance  to  heat  or  drying. 


INFLUENZA  177 

Staining  Reactions. — The  bacillus  stains  with  difficulty.  Loffler's 
methylene  blue  is  a  good  stain,  but  a  pale  red  solution  of  carbol  fuchsin 
in  water  is  better.  The  preparation  must  be  stained  five  to  ten  minutes ; 
if  stained  for  a  shorter  time  or  with  other  stains  the  middle  portion  of 
the  rod  is  often  lighter  coloured  than  the  end  portions.  The  results 
with  the  Gram  method  are  negative. 

Biological  Characters. — The  influenza  bacillus  is  strongly  aerobic, 
and  grows  only  in  the  presence  of  haemoglobin  or  leucocytes.  These 
latter  conditions  explain  why  the  cultivation  of  the  influenza  bacillus  was 
so  long  a  failure. 

Pfeiffer  was  able  sometimes  to  obtain  cultures  from  lung  pus  direct 
on  agar,  but  at  other  times  it  was  impossible.  The  cause  of  the  irregu- 
larity was  that  the  rods  in  the  first  cultures  developed  when  a  trace  of 
blood  was  inoculated  with  the  material,  the  growth  being  negative 
when  no  blood  was  present,  the  same  peculiarity  applying  to  all  the 
daughter  cultivations.  The  influenza  bacillus  can  be  cultivated  regularly 
and  transferred  through  several  generations  in  nutrient  media  containing 
blood.  Blood  agar  is  the  best  medium.  To  obtain  pure  cultures 
Pfeiffer  employs  the  following  method : — 

The  bronchial  sputum  or  exudate  from  the  broncho-pneumonic 
infiltrated  portion  of  the  lung  in  influenza-pneumonia  is  thoroughly 
emulsified  with  1  to  2  c.c.  of  bouillon.  Several  platinum  loops  of  the 
bouillon  are  inoculated,  and  thoroughly  spread  over  the  whole  surface  of 
blood  agar  media,  and  at  the  same  time  control  cultures  are  instituted  on 
ordinary  glycerine  agar  medium. 

The  dilution  of  the  bouillon  has  the  effect  of  segregating  influenza 
bacilli,  so  that  they  grow  in  separate  colonies  on  the  blood  agar  medium, 
while  any  haemoglobin  present  in  the  original  material  is  so  thoroughly 
diluted,  that  the  influenza  bacillus  cannot  develop  on  the  control  agar 
tubes  containing  no  blood. 

The  inoculated  tubes  are  placed  in  the  incubator,  and  in  twenty-four 
hours  the  influenza  colonies  appear  on  the  surface  of  the  blood  agar 
medium  as  closely  compressed  transparent  drops.  The  control  tubes 
are  either  sterile  or  contain  colonies  of  streptococci,  diplococci,  or  other 
bacteria  associated  with  the  influenza  bacillus  in  the  original  material. 

The  transparent  drops  of  the  influenza  colonies  are  mostly  so  small 
that  they  can  only  be  distinctly  seen  with  a  hand  lens.  They  possess  a 
slight  tendency  to  become  confluent,  and  when  closely  arranged  they 
coalesce  into  large  curved  limited  drops,  the  individual  arrangement  of 
the  colonies  being  still  apparent.  When  the  colonies  are  widely  separ- 
ated from  each  other,  they  sometimes  develop  as  large  as  a  pin-head, 
and  still  retain  their  glassy  transparent  appearance.  The  condensation 
water  in  the  tubes  remains  usually  clear,  except  when  it  is  mixed  with 
blood  that  has  fallen  down  the  oblique  surface  of  the  medium,  then 
delicate  white  flakes  develop  in  it. 

M 


178  SPECIAL  BACTERIOLOGY 

In  Bouillon  mixed  with  blood  and  spread  out  in  a  thin  layer  the 
growth  is  somewhat  abundant. 

Plate  Cultures  are  useful  for  the  isolation  of  the  influenza  for  diagnostic 
purposes.  A  little  blood  is  added  to  the  liquefied  agar  before  it  is 
inoculated,  or  if  Petri-dishes  are  used  the  agar  is  allowed  to  set  and  some 
blood  spread  on  the  surface,  and  several  stroke  cultures  made  with  the 
diluted  sputum.  The  developing  colonies  have  the  same  appearance  as 
those  in  the  agar  tubes. 

The  optimum  temperature  for  the  growth  of  the  influenza  bacillus  is 
37°  to  38°  C.,  the  maximum  limit  is  about  42°  C.,  while  the  minimum 
temperature  is  26°  to  27°  C.  At  room  temperature  no  growth  takes 
place.  Oxygen  is  always  necessary  for  the  growth  of  the  influenza 
bacillus,  while  in  presence  of  hydrogen  and  CO2  with  the  addition  of 
blood  to  the  media  no  growth  is  manifest.  PfeifFer  found  that  when  he 
used  blood  serum  or  blood  fibrin  instead  of  blood  the  results  were 
negative,  and  in  further  experiments  he  found  that  haemoglobin  was 
the  necessary  factor  in  the  development  of  the  influenza  bacilli,  as 
haemoglobin  agar  was  just  as  good  a  medium  as  blood  agar.  PfeifFer 
also  obtained  positive  results  with  the  blood  of  rabbits,  guinea-pigs, 
pigeons,  and  fish,  the  growth  with  the  pigeon  blood  being  more 
luxuriant  and  quicker  than  with  human  blood,  owing  to  pigeon's  blood 
being  very  rich  in  haemoglobin. 

Vitality.  —  Heated  to  60°  C.  the  influenza  bacilli  die  in  a  few 
minutes.  At  43°  C.  they  cease  to  grow ;  they  are  not  killed,  for  when 
the  tubes  are  again  placed  at  37°  C.  they  again  develop  colonies.  In 
non-sterile  drinking  water  the  bacilli  die  in  from  twenty-four  to  thirty- 
six  hours.  On  blood  agar  and  in  bouillon  they  live  for  fourteen  to 
eighteen  days,  and  in  moist  sputum  they  appear  to  retain  their 
infectivity.  They  do  not  resist  drying  very  well.  In  blood  or  sputum 
dried  at  37°  they  are  killed  in  one  to  two  hours,  and  at  room  temperature 
in  thirty-six  to  forty  hours. 

Pathogenesis. — PfeifFer  experimented  with  mice,  rats,  guinea-pigs, 
rabbits,  swine,  cats,  dogs,  and  monkeys ;  and  only  in  monkeys  was  he 
able  to  produce  a  disease  simulating  influenza,  by  inoculating  them 
through  the  chest  wall  direct  into  the  lungs,  and  also  by  what  is  a  more 
natural  infection  in  one  monkey,  viz.,  by  introduction  of  the  influenza 
culture  into  the  nose.  The  disease  manifested  itself  with  fever  and 
slight  coughing  for  several  days  ;  an  increase  of  the  inoculated  bacilli  did 
not  occur.  The  introduction  of  large  doses  kills  rabbits  somewhat 
quickly,  the  temperature  falling  rapidly  before  death  ;  it  appears  in  those 
cases  to  act  as  a  poison,  intoxication  symptoms  being  manifested.  Great 
numbers  of  bacilli  are  found  in  rabbits  inoculated  intravenously.  Dead 
cultures  mixed  with  chloroform  act  as  a  strong  poison,  a  fact  that  tends 


BACILLI  OF  PSEUDO-INFLUENZA  179 

to  elucidate  the  cause  of  the  nervous  phenomena  frequently  observed  in 
cases  of  influenza  in  man. 

Immunity. — In  Pfeiffer's  experiments  monkeys  did  not  react  so 
strongly  to  a  second  injection  of  influenza  bacilli  as  they  did  to  the 
first ;  this  fact  he  considered  an  indication  of  immunity.  Man  can 
with  certainty  be  attacked  several  times  with  influenza,  sometimes 
the  same  individual  during  the  course  of  one  epidemic.  It  follows, 
then,  that  in  man  there  is  no  immunity  resulting  from  an  attack  of 
influenza  ;  any  such  condition  can  only  be  considered  as  temporary  and 
only  lasting  a  very  short  time. 

BACILLI    OF    PSEUDO-INFLUENZA. 

In  a  broncho -pneumonic  centre  in  a  diphtheritic  child  PfeifFer  found 
a  bacillus  which  in  appearance  and  staining  reaction  resembled  the 
influenza  bacillus,  and  also  grew  in  a  similar  manner  on  blood  agar. 
Similar  bacilli  have  also  been  isolated  by  other  investigators  in  otitis 
media  and  influenza.  PfeifFer  considers  they  are  allied  to  the  influenza 
bacillus,  and  designates  them  pseudo-influenza  bacilli.  They  are  dis- 
tinguished from  the  genuine  influenza  bacilli  by  their  growth  on  culture 
media,  being  much  more  pronounced  in  all  dimensions  in  twenty-four 
hours,  and  by  their  tendency  to  form  long  false  filaments,  a  condition 
rarely  occurring  in  cultures  of  the  genuine  bacilli. 


The  Septicaemia  Hsemorrhagica  Group  of  Bacteria. 
BACILLUS   BOVISEPTICUS. 

(Ger.  Bac  der  Wild  und  Rinder-seuche.) 

This  disease  occurs  in  two  forms :  one  form  is  characterised  by 
oedema  of  the  skin  and  subcutaneous  tissue — particularly  of  the  head 
— and  swelling  of  the  tongue  ;  the  second  or  pectoral  form  is  character- 
ised by  pleuro-pneumonia,  swelling  of  the  interstitial  tissue  of  the  lungs, 
pleuritis  and  pericarditis.  Both  forms  generally  run  into  haemorrhagic 
enteritis. 

The  mortality  is  about  90  per  cent. 

The  Wild  and  Rinder-seuche  is  mentioned  as  identical  with  the 
Schweine-seuche,  because  experimentally  the  bacillus  of  Schweine-seuche 
was  found  pathogenic  for  calves,  and  the  Rinder-seuche  likewise  patho- 
genic for  swine  in  several  instances. 

The  bacillus  was  first  observed  by  Kitt,  and  further  studied  by  Kitt 
and  Hueppe,  and  classified  as  one  of  the  septicaemia  haemorrhagica 
group  of  organisms.  The  Italian  buffalo  disease  (Barbone  dei  bufali) 


180  SPECIAL  BACTERIOLOGY 

is  probably  identical  with  the  Rinder-seuche,  as  the  Italian  investigator 
found  an  organism  very  similar  to  that  of  the  Schweine-seuche,  with 
which  they  inoculated  the  following  animals  with  positive  results :  a 
young  buffalo,  a  young  pig,  a  young  horse,  a  young  cow,  a  sheep,  also 
mice,  rats,  rabbits,  guinea-pigs,  pigeons,  and  chickens. 


SEPTIC   PLEURO-PNEUMONIA   OF   CALVES. 

This  disease  occurs  in  certain  localities  in  an  enzootic  form,  attacking 
very  young  calves,  the  affected  animals  dying  very  quickly.  The 
lesions  produced  are  somewhat  analogous  to  those  of  pleuro-pneumonia 
contagiosa,  but  the  thickening  of  the  interlobular  connective  tissue  is 
less  marked,  and  the  exudation  of  lymph  not  so  abundant.  There  is 
also  a  want  of  uniformity  of  colour  in  the  individual  pulmonary  lobules 
in  some  cases.  Poels  found  sero-fibrinous  exudates  and  pleuritic  ad- 
hesions frequent.  Vanden  also  mentions  the  occasional  occurrence  of 
inflammation  of  the  pericardium,  liver,  kidneys,  stomach  and  intestines. 

Microscopical  Appearances. — In  the  lung  and  muco-pus  of  the 
bronchi  small  ovoid  organisms  are  present,  with  rounded  ends  1  //,  to 
1-5  /A  long,  and  0*5  //,  thick. 

Staining  Reactions. — Stain  easily  with  the  ordinary  aniline  stains, 
but  not  by  Gram's  method.  When  weak  solutions  of  gentian  violet  are 
used,  they  exhibit  the  bipolar  staining  characteristic  of  the  septicaemia 
haemorrhagica  group  of  bacteria. 

Motility. — Very  motile. 

Biological  Characters. — Grows  well  in  bouillon  and  on  solid  media. 

Pathogenesis. — Rabbits  die  in  twenty-four  to  forty-eight  hours 
either  by  ingestion  or  inoculation.  Intrapulmonary  injection  of  1 
drop  produces  pneumonia.  Calves  also  died  by  intrapulmonary 
injection ;  sheep  and  dogs  are  immune.  It  is  a  facultative  parasite 
capable  of  living  in  the  soil,  which,  according  to  Poels,  explains  the 
presence  of  the  disease  on  an  infected  farm. 


BACILLUS  DYSENTERIC  VITULORUM. 

(Bacillus  of  White  Diarrhoea  of  Calves — Jensen.) 

Described  by  Jensen  as  the  cause  of  the  so-called  white  diarrhoea 
or  scour  occurring  amongst  calves. 

Microscopical  Appearances. — Small  bacilli  a  little  larger  than  the 
chicken  cholera  bacillus. 

Motility. — Non-motile. 


BACILLUS  CHOLERA  COLUMBARUM  181 

Staining  Reactions. — Exhibits  polar  staining  with  the  ordinary 
reagents.  By  the  Gram  method  the  reaction  is  negative. 

Biological  Characters. — The  growth  is  luxuriant  on  the  usual 
media,  and  very  similar  to  the  B.  coli  communis. 

On  Potatoes  it  forms  a  brown-coloured  shiny  growth ;  the  cultures 
give  off  an  unpleasant-smelling  gas. 

Pathogenesis. — When  new-born,  or  very  young  calves  are  fed  with 
5  c.c.  of  a  bouillon  culture,  a  deadly  diarrhoea  is  produced,  death  occurring 
in  the  course  of  one  to  two  days,  the  bacilli  being  found  in  the  intestines 
and  interior  of  the  organs.  In  sections  they  were  observed  in  clusters 
in  the  small  bloodvessels. 


BACILLUS  CHOLEILE  COLUMBARUM. 

This  organism  was  found  by  Leclainche  in  an  epidemic  among 
wild  pigeons,  and  is  probably  a  variety  of  the  fowl  cholera  bacillus. 

Microscopical  Appearances. — Similar  to  those  of  the  fowl  cholera 
bacillus,  but  a  little  larger. 

Motility. — Non-motile. 
Spore  Formation  absent. 

Staining  Reactions.  —  On  Gelatine  and  A  gar  Media  growth  is 
similar  to  fowl  cholera  bacillus. 

Bouillon  is  not  clouded,  but  a  flaky  sediment  is  formed. 
On  Potatoes  at  20°  C.  it  forms  a  greyish-yellow  layer. 

Pathogenesis. — Wild  pigeons  are  most  susceptible,  dying  in  three 
to  six  days  by  feeding  ;  and  in  two  days,  when  injected  intravenously,  the 
symptoms  manifested  are  drowsiness,  diarrhoea,  convulsions,  and  septi- 
caemia. The  tame  pigeon  is  not  so  liable  to  infection.  Fowls  are 
immune,  also  dogs  and  cats.  Rabbits  die  in  about  eight  days,  and 
guinea-pigs  in  about  ten  days,  when  subcutaneously  inoculated. 


BACILLUS  OF  CHOLERA  IN  DUCKS. 

This  organism  was  found  by  Cornil  and  Toupet  in  an  epidemic 
among  the  ducks  in  the  Jardin  d' Acclimation  at  Paris.  The  disease 
was  characterized  by  diarrhoea,  feebleness,  and  muscular  tremors, 
resulting  fatally  in  two  to  three  days. 

Microscopical  Appearances. — Morphologically  identical  with  the 
bacillus  of  fowl  cholera. 


182  SPECIAL  BACTERIOLOGY 

Motility . — N  on-motile . 
Spore  Formation  absent. 

Staining  Reactions. — Exhibits  the  usual  bipolar  staining  with 
the  ordinary  aniline  stains.  Does  not  stain  by  the  Gram  method. 

Cultivation. — It  is  an  aerobic,  non-liquefying  bacillus,  growing  in 
the  usual  culture  media  at  room  temperature.  The  growth  in  the 
various  media  corresponds  to  that  of  the  bacillus  of  fowl  cholera. 

Pathogenesis. — Affects  ducks,  but  not  chickens  or  pigeons,  and 
only  kills  rabbits  when  injected  in  large  quantities.  Ducks  die  in  one 
to  three  days  from  subcutaneous  inoculations,  or  by  ingestion  of  food 
containing  the  bacillus. 


BACILLUS   OF   FOWL   CHOLERA. 

(Ger.  Bac  der  Huhner  Cholera  ;  FT.   Cholera  des  poules). 

This  disease  often  occurs  in  poultry  as  an  epizootic ;  it  is  char- 
acterized by  diarrhoea  and  death  in  one  to  two  days. 

Microscopical  Appearances. — Very  short  bacilli  of  varying  size. 
Motility. — Non-motile. 
Spore  Formation  absent. 

Staining  Reactions. — In  cover-glass  specimens  the  bacilli  stain 
with  the  ordinary  aniline  dyes,  more  at  the  pole  than  in  the  middle, 
giving  them  the  appearance  of  diplococci  (see  Photomicrograph,  Fig.  66), 
but  by  intensive  staining  they  appear  as  genuine  bacilli.  By  the  Gram 
method  the  bacilli  are  decolorized. 

Vitality. — Exhibit  slight  resistance  to  heating  and  drying,  but 
remain  lying  a  long  time  in  contaminated  or  mixed  cultures. 

Biological  Characters. — The  bacilli  grow  on  the  ordinary  media 
at  both  room  and  incubator  temperature. 

On  Gelatine  Plates,  in  the  deep  portions  of  the  medium,  they  form 
round,  irregular,  brownish  discs,  and  on  the  surface  the  growth  is  slow 
and  limited. 

In  Gelatine  Stab  Cultures  the  growth  occurs  on  the  surface,  as  well  as 
along  the  course  of  the  needle.  The  surface  growth  consists  of  a 
delicate  greyish-white  coating. 

On  Agar  and  Blood  Serum  Media  a  glistening  whitish  coating  is 
formed. 

On  Potatoes  it  does  not  grow  at  ordinary  temperature,  but  at  higher 
temperatures  a  transparent,  greyish -white,  flat  coating  is  formed. 


Fio.%65.— Bacillus  of  Influenza  in  Sputum.    Methylene 
blue.     X  1000. 


FIG.  6C.— Bacillus  of  Fowl  Cholera.    Cover-glass  specimen  fron 
inoculated  mouse.    Fuchsin.     X  1000. 


[T.  BowJiill,  F.H.C.V.S.,  Photo.,  Edinburyli,  1SOS. 


BACILLUS  OF  FOWL  CHOLERA  183 

Bouillon  is  slightly  clouded. 

Milk  is  gradually  coagulated  by  the  formation  of  an  acid. 

Litmus  is  reduced.     Indol  and  Phenol  are  formed. 

Pathogenesis. — The  bacilli  cause  a  typical  septicaemia  in  small 
doses  by  cutaneous  inoculation  (and  in  larger  doses  by  feeding — Giinther) 
in  pigeons,  fowls,  geese,  ducks,  pheasants,  small  birds,  also  birds  of  prey, 
rabbits,  and  mice.  Guinea-pigs,  sheep,  and  horses  exhibit  only  a  local 
reaction ;  suppuration  at  the  point  of  inoculation.  Dogs  and  cats  can 
consume  large  quantities  of  the  cadavers  of  infected  animals  without 
becoming  sick.  Man  can  also  tolerate  infected  meat. 

The  bacilli  are  present  in  enormous  quantities  in  the  bloodvessels  of 
affected  animals.  In  pigeons,  and  especially  in  chickens,  the  point  of 
inoculation  is  greatly  inflamed,  tending  to  necrosis.  In  the  intestines  a 
hsemorrhagic  enteritis  occurs.  Chickens  and  rabbits  exhibit  pneumonic 
lesions.  Pericarditis  and  haemorrhages  on  the  pericardium  are  common. 
The  spleen  and  liver  are  also  enlarged.  The  bacilli  of  fowl  cholera  pass 
from  the  mother  to  the  foetus — that  is,  to  the  eggs. 

Immunity. — Pasteur  found  that  artificial  cultures  of  fowl  cholera 
bacilli,  if  left  exposed  to  the  air  for  a  long  time,  lost  their  power  of 
killing  chickens.  The  inoculated  birds  were  only  locally  affected,  and 
afterwards  were  immune  to  infection  with  virulent  cultures. 


BACILLUS   PHASIANI   SEPTICUS. 

This  organism  was  discovered  by  E.  Klein  in  an  epizootic  amongst 
young  pheasants.  The  author  isolated  a  morphologically  identical 
organism  in  an  outbreak  amongst  some  young  pheasants,  the  mortality 
being  very  great.  The  principal  lesions  were  catarrhal  inflammation 
of  the  bowels  and  enlargement  of  the  liver,  with  slight  broncho- 
pneumonia. 

Microscopical  Appearances. — Small  bacilli,  very  like  the  Bacillus 
coli  communis,  but  smaller  and  shorter  (see  Photomicrograph,  Fig.  67). 
Motility .  —Strongly  motile. 
Spore  Formation  absent. 

Staining  Reactions.— Easily  stained  with  any  of  the  ordinary 
aniline  dyes,  but  not  by  the  Gram  method. 

Biological  Characters. — Similar  to  those  of  Bacillus  coli  communis, 
except  that  milk  is  not  coagulated. 

Pathogenesis.— In  the  natural  course  of  the  disease,  death  takes 
place  among  young  pheasants  in  a  few  days  or  within  a  week.  Young 
pheasants  are  killed  in  twenty-four  hours  with  a  few  drops  of  a  bouillon 


184  SPECIAL  BACTERIOLOGY 

culture,  with  symptoms  of  drowsiness  and  stupor.  Diarrhoea  is  an 
inconstant  symptom ;  septicaemia  also  occurs.  Young  chickens,  pigeons, 
rabbits,  and  guinea-pigs  do  not  die  when  injected  with  J  c.c.  of 
bouillon  culture. 

It  is  distinguished  from  the  bacillus  of  chicken  cholera  by  its  motility 
and  its  slight  pathogenic  action  on  the  most  of  animals,  and  the  fact 
that  the  bacillus  of  chicken  cholera  coagulates  milk. 

BACILLUS   OF   THE   GROUSE   DISEASE. 

This  is  an  infectious  disease  affecting  red  grouse,  due  to  a  bacillus 
discovered  by  E.  Klein.  The  affected  animals  exhibit  pneumonic 
lesions  ;  the  mucosa  and  serosa  of  the  intestines  are  congested ;  the 
liver  is  also  congested  and  dark  coloured.  The  bacilli  are  present  in 
the  bloodvessels  and  extravasated  blood,  and  sometimes  in  the  heart's 
blood. 

Microscopical  Appearances. — Bacilli  0'4>  by  0-6  by  1*6  /M,  oval  or 
coccus-like,  and  sometimes  a  few  are  rod-shaped. 

Motility. — Motile  in  recent  cultures.  When  some  days  old,  only  a 
few  of  the  bacilli  are  motile. 

Spore  Formation  absent. 

Staining  Reactions. — Stains  with  the  ordinary  stains,  but  not  by 
the  Gram  method. 

Biological  Characters. — On  Gelatine  Plates  the  surface  colonies  are 
irregular,  and  the  deep  colonies  are  small  and  round. 

In  Stab  Cultures. — Nail-like  culture  with  flat  head. 

On  Agar. — A  thin  greyish  coating. 

Bouillon  becomes  clouded. 

In  Grape  Sugar  Media  gas  is  formed. 

Pathogenesis. — Very  virulent  for  mice,  and  not  so  virulent  for 
guinea-pigs,  by  subcutaneous  injection.  The  bacilli  soon  lose  their 
virulence,  but  soon  regain  it,  when  cultivated  in  bouillon  to  which  some 
small  pieces  of  hard-boiled  egg  albumen  is  added.  The  yellow-hammer 
and  finch  are  easily  affected  by  subcutaneous  injection,  while  sparrows 
are  not  so  susceptible.  Feeding  experiments  yield  no  positive  results. 
Infection  probably  by  means  of  the  air. 

BACILLUS  OF  THE  CANARY  BIRD  SEPTICAEMIA. 

Found  by  Von  Rieck. 

Microscopical  Appearances.— Bacilli  somewhat  larger  than  the 
bacillus  of  chicken  cholera,  1-2  to  2- 5  //,  long. 


BACILLUS  OF  CANARY  BIRD  SEPTIC^MIA  185 

Motility.— Motile. 

Staining  Reactions. — Stains  with  the  ordinary  stains,  most  in- 
tensely at  the  poles.  Not  by  the  Gram  method. 

Biological  Characters. — The  growth  on  the  various  media  is  more 
luxuriant  than  the  bacillus  of  chicken  cholera. 

On  Potatoes  it  forms  a  yellow-grey  coating. 

Vitality. — The  cultures  are  killed  when  heated  for  five  minutes 
at  100°  C. 

Pathogenesis. — Mice  die  when  inoculated  cutaneously  or  sub- 
cutaneously  with  minute  quantities,  and  exhibit  a  characteristic  sep- 
ticaemia. Feeding  experiments  yield  similar  results.  Affected  canaries 
exhibit  a  fuliginous  colouring  of  the  skin  and  multiple  necrosis  of  the 
liver.  Bacilli  are  present  in  great  quantities  in  the  blood. 

Differential  Diagnosis. — From  chicken  cholera  it  is  distinguished 
by  the  motility  of  the  organism  and  its  growth  on  potatoes. 

BACILLUS  OF  PNEUMONIA-PERICARDITIS  OF  THE 
TURKEY  (M'FADYEAN.) 

This  organism  was  described  by  M'Fadyean  in  an  epizootic  among 
turkeys  characterised  by  pneumonia-pericardial  lesions. 

Microscopical  Appearances. — In  the  blood  tissues  and  artificial 
culture  media  it  occurs  as  short  ovoid  bacilli,  not  distinguishable  by 
shape  or  size  from  the  bacilli  of  fowl  cholera. 

Motility.— Motile. 

Spore  Formation  absent. 

Staining  Reactions. — Exhibits  bipolar  staining  with  the  ordinary 
aniline  dyes,  but  is  decolorized  by  the  Gram  and  Weigert  methods. 

Cultivation. — Stab  Cultures  in  Gelatine  kept  at  25°  C.  exhibit  a 
distinctly  visible  growth  in  forty-eight  hours  along  the  needle  track. 
The  gelatine  is  not  liquefied. 

In  Streak  Cultures  in  Oblique  Gelatine  it  develops  in  a  whitish  line, 
which  does  not  spread  far  from  the  needle  track. 

On  oblique  surface  Agar  at  the  temperature  of  the  body  it  develops  a 
thin  translucent  pellicle. 

Bouillon  becomes  turbid  in  twenty-four  hours  at  37°  C.,  a  ropy  sedi- 
ment finally  forming,  the  upper  portions  of  the  bouillon  becoming 
clear. 

On  Potatoes  no  appreciable  growth  takes  place. 

The  bacillus  is  a  facultative  anaerobe,  growing  abundantly  in 
bouillon  flasks  in  an  atmosphere  of  hydrogen. 


186  SPECIAL  BACTERIOLOGY 

Pathogenesis.— The  results  with  guinea-pigs  and  rabbits  are 
similar  to  those  obtained  with  the  bacillus  of  fowl  cholera,  very  virulent 
for  the  rabbit,  but  not  so  virulent  for  the  guinea-pig ;  infection  by 
feeding  did  not  succeed.  Fowls  and  pigeons  become  slightly  affected. 
Other  birds  were  not  affected.  A  calf  and  a  pony  manifested  only 
slight  lesions  at  the  point  of  inoculation.  The  disease  was  produced  in 
turkeys  inoculated  with  pure  cultures,  the  symptoms  being  stiffness, 
weakness,  nasal  catarrh,  rattling  in  the  throat,  and  then  milk-white 
evacuations.  The  autopsy  revealed  pneumonia  and  pericarditis.  In  the 
lungs  and  other  organs  great  numbers  of  the  bacilli  were  present.  The 
bacillus  is  differentiated  from  that  of  fowl  cholera  by  being  motile,  by 
its  slight  action  in  other  birds,  and  localization  in  the  lungs. 

PNEUMO-ENTERITIS   OF  THE   SHEER 

According  to  M.  Galtier,  this  disease  in  sheep  is  caused  by  the  same 
organism  as  swine  fever  or  hog  cholera.  It  sometimes  occurs  in  an 
epizootic,  causing  great  ravages  in  affected  flocks.  Outbreaks  have  been 
observed  when  recently  purchased  swine,  that  had  contracted  swine 
fever  in  the  market  pens,  have  been  placed  in  sheep  folds.  When 
once  established,  the  disease  transmits  itself  with  great  rapidity  from 
sheep  to  sheep,  and  it  is  more  severe  and  more  frequently  fatal  in 
young  animals.  The  general  symptoms  are  lassitude,  general  loss  of 
vigour,  high  fever,  and  loss  of  rumination ;  these  symtoms  are  soon 
followed  by  tymphany,  foetid  and  exhaustive  diarrhoea,  quickened  re- 
spirations, mucous  discharge  from  the  nostrils  sometimes  tinged  with 
blood,  and  the  special  symptoms  of  broncho-  and  pleuro-pneumonia. 
The  skin  and  visible  mucous  membranes  present  a  more  or  less  vivid 
red  colour,  sometimes  mixed  with  hsemorrhagic  spots.  Abortion  is 
often  observed,  although  the  mother  does  not  necessarily  succumb  to 
the  disease.  The  intensity  of  the  disease  varies ;  it  is  sometimes  so 
severe  that  death  occurs  in  a  few  hours  or  days,  and  again  the  attack 
may  be  so  slight  as  to  be  hardly  perceptible.  Convalescence  from  the 
severe  forms  is  always  prolonged. 

M.  Galtier  further  states  that  pneumo-enteritis  (or  swine  fever), 
which  is  generally  considered  to  be  peculiar  to  the  pig,  extends  to  all 
farm  animals,  especially  to  the  sheep,  bovines,  and  solipeds.  The  disease 
being  transmitted  to  the  foetus,  calves  coming  from  diseased  cows  which 
are  or  have  been  subject  to  coughing,  are  born  with  the  germ  of  the 
disease  in  them,  and  die  in  a  few  days  with  the  lesions  of  broncho- 
pneumonia  and  enteritis  (pneumo-enteritis  of  calves).  Whether  this 
conclusion  is  correct  or  justified  is  largely  open  to  question,  as  though 
the  morphology  of  the  bacilli  of  the  septicaemic  group  are  very  similar, 
the  predilection  of  the  different  organisms  for  different  animals  is  widely 
diverse. 


BACILLUS  FELIS  SEPTICUS  187 


BACILLUS   FELIS   SEPTICUS. 

This  bacillus  was  isolated  regularly  by  Fiocca  from  the  saliva  of 
cats. 

Microscopical  Appearances. — Very  small  short  rods,  0-2  to  0-3  //, 
thick,  often  occurring  as  diplococci. 

Spore  Formation  absent. 

Staining  Reactions.  —  By  ordinary  methods,  but  not  by  the 
Gram  method. 

Biological  Characters. — Its  growth  is  similar  to  the  rabbit 
septicaemia  bacillus. 

In  Bouillon  it  forms  no  flakes. 

Milk  is  not  coagulated. 

On  Potatoes  a  very  thin,  almost  invisible  coating. 

Sugar  Media  is  not  fermented. 

Pathogenesis. — Produces  septicaemia  in  mice,  rabbits,  guinea-pigs, 
and  young  rats. 

BACILLUS  TYPHI  MURIUM. 

(Mouse  Typhoid.) 

This  bacillus  was  discovered  by  Loffler  in  an  epidemic  among 
mice. 

Microscopical  Appearances. — Small  rods,  which  often  form  long 
threads. 

Motility. — Strongly  motile. 

Staining  Reactions. — Easily  stained  with  the  ordinary  stain,  but 
not  by  the  Gram  method. 

Biological  Characters. — On  Gelatine  Plates  the  deep  colonies  are 
small,  round,  slight,  granular,  and  of  yellowish-brown  colour  ;  the  super- 
ficial colonies  are  flattened,  irregularly  notched,  and  possess  delicate 
furrows  similar  to  the  colonies  of  the  typhoid  bacillus,  only  more  granu- 
lar and  of  more  luxuriant  growth. 

In  Stab  Cultures. — Nail-like  growth  with  a  flat  top. 

On  Agar,  Blood  Serum,  and  Bouillon  the  growth  exhibits  no  special 
characteristics. 

On  Potatoes  a  whitish,  not  specially  luxuriant  coating  forms,  the 
surrounding  medium  being  stained  a  dirty  greyish-blue. 


188  SPECIAL  BACTERIOLOGY 

In  Bouillon  containing  sugar,  gas  is  formed. 
Milk  is  not  coagulated. 

Pathogenesis. —  Very  virulent  for  white  and  grey  house  mice,,  and 
for  field  mice  (arvicola  arvalis)  both  by  subcutaneous  injection  and  by 
feeding.  The  infection  is  spread  by  the  living  mice  eating  the  bodies 
of  those  dead  of  the  disease.  Loffler,  owing  to  this  circumstance,  used 
the  cultures  in  destroying  the  mice  during  the  plague  in  Thessaly. 

SWINE   FEVER. 

(Pneumo-enteritis — Klein.       Ger.  Bacillus  der  Schweine  Pest. 
America,  Swine  Plague — Billings;  Hog  Cholera — Smith). 

The  chief  veterinary  officer,  in  his  report  to  the  Board  of  Agri- 
culture for  1896,  states: — ''It  is  quite  certain  that  the  disease  which 
exists  among  the  swine  in  America,  where  it  has  received  the  name  of 
hog  cholera,  is  identical  with  our  swine  fever,  because  in  the  year  1879 
some  cargoes  of  pigs  affected  with  hog  cholera  were  landed  at  Liverpool, 
when  an  opportunity  was  afforded  of  identifying  the  lesions  of  that 
disease  with  swine  fever? 

The  above  conclusions  are  identical  with  those  published  by  the 
writer  in  1891,  when  cultures  were  obtained  from  an  outbreak  of 
swine  fever  in  England,  and  sent  to  Billings  in  America,  who  wrote 
that  he  had  made  all  the  tests  and  control  experiments,  concluding  as 
follows : — '  It  is  the  same  germ  as  we  have  here  as  the  cause  of  hog 
cholera."1 

Microscopical  Appearances. — Short  bacilli  1-2  to  1-5  /*  long,  and 
0-6  to  0-7  p  broad. 

Motility. — Strongly  motile ;  possessing  a  multitude  of  flagella, 
something  like  the  Bacillus  typhi  abdominalis  peritricha  (see  Photo- 
micrograph, Plate  III.,  Fig.  13). 

Staining  Reactions. — Stain  easily  with  any  of  the  usual  aniline 
dyes.  The  Gram  method  gives  negative  results.  When  freshly  obtained 
from  an  animal,  the  condition  known  as  bipolar  staining  is  well-marked 
(see  Photomicrograph,  Figs.  68,  69,  70),  the  clear  unstained  centres 
not  being  so  easily  differentiated  in  older  cultures,  or  if  the  staining 
process  is  too  long  continued.  In  sections  of  organs  the  bacilli  are 
present  in  the  capillaries  and  small  veins. 

Spore  Formation  absent. 

Vitality. — It  is  destroyed  by  a  temperature  of  58°  C.  in  from  fifteen 
to  twenty  minutes.  It  preserves  its  vitality  in  spite  of  desiccation  for 
nearly  two  months,  and  vegetates  and  multiplies  at  the  ordinary  tern- 


Fio.  67.— 


B.  Phasiani  Septicus.    Cover-glass  specimen.    Agar  culture. 
Fuchsin.     X  1000. 


FIG.  68.— Bacillus  of  Swine  Fever.    Spleen  of  pig.    Methyleno  blue. 
X  1000. 


[T.  Boivhill,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


SWINE  FEVER  189 

perature  of  summer,  and  retains  its  vitality  for  more  than  fifteen  days 
in  sterilized  water. 

Biological  Characters. — On  Gelatine  Plates  the  surface  colonies 
are  spread  out  flat  with  round  or  irregular  borders ;  the  deeper  colonies 
are  small,  almost  homogeneous  brownish  spheres. 

Stab  Cultures  in  Gelatine  resemble  a  nail  with  a  flat  top  (see  Photo- 
micrograph, Fig.  73.) 

On   Agar-Agar   Media   a   greyish-white    transparent   growth  occurs, 
and  in  Grape  Sugar  A  gar  gas  is  formed. 
On  Potatoes  a  yellowish  growth  occurs. 
Bouillon  becomes  cloudy,  and  a  film  forms  on  the  surface. 
Milk  remains  unchanged,  and  the  reaction  alkaline. 
Neither  Indol  nor  Phenol  are  formed. 

Pathogenesis. — A  small  quantity  of  a  culture  kills  rabbits  and  mice 
in  seven  to  twelve  days  with  elevation  of  temperature.  The  lesions  found 
are  enlarged  spleen  of  a  dark  red  colour,  and  necrotic  patches  in  the 
liver.  The  kidneys  are  inflamed,  and  the  urine  contains  albumen.  The 
substance  of  the  heart  is  flabby,  and  fatty  degeneration  is  present.  The 
mucosa  of  the  small  intestine  is  swollen,  and  the  contents  are  shiny,  and 
ecchymoses  are  often  present  here  and  in  the  duodenum.  The  charac- 
teristic bacilli  are  present  in  small  masses  in  all  the  organs,  mostly  in 
the  capillaries  and  small  veins.  Injection  of  the  virus  into  the  lungs 
causes  pneumonic  changes.  Feeding  with  cultures  and  inhalation  also 
cause  infection.  Pigeons  are  somewhat  refractory,  but  can  be  affected 
by  large  doses.  Chickens  are  not  affected  even  with  large  quantities  of 
the  virus.  Swine  are  somewhat  refractory  to  subcutaneous  injections, 
but  by  intravenous  injection  of  1  to  2  c.c.,  and  feeding  with  cultures,  a 
severe  diphtheric  inflammation  of  the  stomach  and  large  intestines  occurs. 
In  other  cases  the  bacilli  are  less  virulent,  especially  when  obtained 
from  chronic  cases  of  swine  fever.  The  characters  of  the  cultures  can, 
moreover,  also  vary.  The  necrosis  of  the  liver  is  not  present  in  the 
milder  forms  of  the  disease,  therefore  the  changes  in  the  bowel  are 
more  pronounced.  Abscesses  sometimes  develop  under  the  skin  at  the 
point  of  inoculation.  Swine  fever  sometimes  occurs  in  devastating 
epizootics,  especially  in  America,  when  90  per  cent,  of  the  swine  may 
die.  It  occurs  in  an  acute  or  haemorrhagic  septicaemic  form,  killing  the 
animal  in  a  few  days ;  and  in  a  chronic  form,  when  the  disease  may  last 
two  to  four  weeks,  or  even  longer.  The  post-mortem  examination  of 
affected  animals  presents  a  variety  of  lesions,  which  vary  according  to 
the  severity  and  length  of  time  the  animal  has  been  affected.  The 
following  is  a  summary  of  lesions  observed  in  cases  of  swine  fever  : — 

Tumefactions  round  the  head  and  back,  the  tongue  darkish  in  colour, 
and  small  necrosed  patches  and  ulcers  are  often  present  on  the  lips, 
gums,  and  tongue.  Along  the  abdomen  numerous  dark  red-blue 


190  SPECIAL  BACTERIOLOGY 

blotches,  terminating  diffusely  in  the  surrounding  tissue.  On  cutting 
through  the  skin,  dark  red  blood  escapes  from  the  cut  bloodvessels. 
On  section  the  abdominal  cavity  usually  contains  a  large  quantity  of 
straw-coloured  lymph  with  numerous  flocculi  floating  in  it. 

The  Large  Intestines  are  sometimes  agglutinated  together  with  bands 
of  flocculent  lymph,  the  adhesions  being  very  resistant  in  some 
parts. 

The  Mesenteric  Lymph  Glands  are  usually  enlarged,  and  on  section 
present  a  greyish-red  striated  appearance.  The  mucosa  of  the  large 
intestines  is  usually  red  and  swollen,  the  ileo-caecal  valve,  or  valve  of 
Bauhini,  swollen  and  often  the  seat  of  extensive  ulcerations.  The 
difference  between  ulceration  and  the  deposition  of  caseous  matter  in 
the  follicle  of  the  glands  in  the  neighbourhood  of  the  valve  must  be 
carefully  noted,  otherwise  an  incorrect  diagnosis  will  result. 

The  Small  Intestines  usually  present  a  reddish  appearance  (typhilitis), 
the  internal  surface  being  studded  with  numerous  dark  red  spots.  The 
mucosa  is  swollen,  with  diffused  capillary  redness,  Peyer's  patches 
enlarged,  and  contents  of  intestines  fluid.  The  characteristic  ulceration 
mentioned  in  connection  with  the  ileo-caecal  valve  may  also  be  present, 
and,  although  involving  the  whole  thickness  of  the  bowel,  perforation  is 
extremely  rare. 

Liver. — This  organ  is  usually  enlarged,  edges  rounded.  On  section 
dark  blood  exudes,  and  the  acini  are  considerably  enlarged.  Occa- 
sional centres  of  necrosis  have  also  been  observed. 

The  Gall  Bladder  is  usually  distended,  and  full  of  viscid,  dark  greenish- 
yellow  gall. 

The  Spleen  is  sometimes  enormously  enlarged  and  the  pulp  slightly 
disintegrated.  Trabeculae  thickened,  the  Malpighian  corpuscles 
enlarged,  and  sometimes  a  few  white  spots  on  the  capsules. 

The  Stomach  is  sometimes  the  seat  of  numerous  ulcerations  (see 
Photo.,  Fig.  71),  and  in  other  cases  the  cardiac  portion  is  healthy,  while 
the  pylorus  is  congested  and  ulcerated. 

The  Kidneys  are  sometimes  enormously  swollen,  and  in  a  state  of 
haematogenous  nephritis,  the  pelvis  in  some  instances  being  almost 
entirely  occluded  with  blood  clots.  Sometimes  the  kidneys  are  in  a 
state  of  parenchymatous  degeneration,  and  small  petechiae  may  also 
be  observed  under  the  capsule,  which  may  or  may  not  be  adherent. 

The  Bladder  sometimes  contains  haematogenous  urine  depending  on 
the  condition  of  the  kidneys. 

The  superficial  and  deep  inguinal  glands  are  usually  in  the  same 
condition  as  those  of  the  mesenteric  and  bronchial  regions. 

Thoracic  Cavity. — It  is  in  this  part  of  the  animal  that  such  a  wide 
difference  of  opinion  exists  among  experts,  as  to  whether  pneumonia  is 
or  is  not  a  diagnostic  symptom  of  swine  fever.  The  following  thoracic 


Kic.  r,'.>.  -Bacillus  of  Swine  Fever.    Piu~e  culture  from  lymph 
gland  of  pig.     Methylene  blue.     X  1000. 


FIG.  70.— Bacillus  of  Swine  Fever.     Pure  culture  from  lymph  gland 
of  pig.    Methylene  blue,     x  1500. 


i  [T.  BoH'liill,  V.R.C.V.S.,  Photo.,  AW /„/,„,•<,//,  18'J.S 


FK;.  71. — JStoiiKicli  from  pi^  Uead  of  svviue  fever,  showing 
ulcerations. 


FKJ.  72. — Broncho-Pneumonia  suis,  lung  of  i»ij^  dead  of  swine  fever. 

[T.  Jioirlii/I,  t'.R.i'.l'.S.,  I'/ioto.,  folhilmrgh,  1898. 


SWINE  FEVER  191 

lesions  have  been  observed  in  genuine  cases  of  swine  fever: — Slight 
effusion  into  the  thorax ;  attachments  or  adhesions  of  the  pleurae  may 
be  present  or  absent.  Small  ecchymosed  spots  are  sometimes  found  on 
the  pleura  costalis  pulmonalis,  and  also  on  the  pericardium. 

The  pericardial  sac  may  be  distended  with  an  enormous  quantity 
of  bright  yellow  fluid,  and  the  visceral  folds  studded  with  numerous 
bright  red  ecchymosed  spots. 

The  Bronchial  Lymph  Glands  are  usually  swollen,  juicy,  and  similar  to 
those  of  the  mesenteric  region. 

The  Lungs  are  sometimes  marked  by  numerous  red  or  reddish  blue- 
black  spots  of  various  sizes,  some  of  which  extend  beyond  the  surface 
of  the  lung,  and  often  correspond  to  a  single  lobulus  ;  large  centres  of 
hepatization  are  also  often  present  (see  Photo.,  Fig.  72).  On  section 
some  of  the  lungs  reveal  a  stage  of  broncho-pneumonia  lobularis,  the 
bronchial  mucosa  being  swollen  ;  and  a  yellowish-red  exudate  is  present 
in  the  bronchi. 

Immunity. — Billings  obtained  protection  against  the  disease  by 
inoculating  swine  with  cultures  derived  from  mild  cases  of  the  natural 
disease,  but  a  certain  proportion  of  the  animals  died  from  the  effects 
of  the  inoculation.  Smith  claims  to  have  obtained  better  results  by 
injecting  small  doses  of  the  virus  intravenously.  Schweinitz  succeeded 
in  vaccinating  the  guinea-pig  by  means  of  soluble  substances,  which 
he  obtained  from  cultures.  The  same  authority  has  recently  pro- 
duced immunity  by  means  of  an  antitoxic  serum  obtained  from  the 
blood  of  immune  animals.  A  number  of  guinea-pigs  were  inoculated 
with  serum  from  a  pig  rendered  immune  to  hog  cholera,  the  doses 
varying  from  0'5  to  4*5  c.c.  Five  of  the  inoculated  guinea-pigs  died 
of  blood-poisoning;  three  withstood  the  injection,  and  fifteen  days 
later  also  resisted  the  otherwise  deadly  injection  of  1-10  c.c.  of  a  one 
day  old  hog  cholera  culture.  In  another  experiment,  three  guinea- 
pigs  eight  months  after  being  immunized  still  remained  capable  of 
resisting  the  above-mentioned  virulent  dose.  The  immunity  did  not 
continue  for  a  longer  period,  because  the  serum  experimented  with 
was  obtained  from  a  pig  immunized  a  long  time  before.  It  therefore 
appears  that  the  antitoxic  substance  is  only  present  free  in  the  blood, 
and  capable  of  conferring  immunity  on  other  animals,  immediately 
after  the  animal  is  immunized  ;  but  when  already  immunized  animals 
receive  further  inoculations  of  virus,  a  new  formation  of  antitoxic 
substance  results.  Schweinitz  also  produced  immunity  against 
Schweine-seuche  with  an  antitoxic  serum,  but  it  was  not  possible 
with  the  Schweine-seuche  serum  to  produce  immunity  against  hog 
cholera,  or  vice  versa. 


192  SPECIAL  BACTERIOLOGY 

BACILLUS  SUISEPTICUS. 

(Schweine-seuche — Schiitz  ;  Swine  Plague — Smith.      Infectious  Pneu- 
monia of  the  Pig.) 

This  bacillus  was  first  described  by  Loffler  and  Schiitz,  and  is  also 
mentioned  by  Smith  as  being  associated  with  his  hog  cholera  germ, 
as  well  as  occurring  alone. 

Microscopical  Appearances. — Short  bacilli  morphologically,  and  in 
cultures  very  like  the  chicken  cholera  bacillus.  (See  Photomicrograph, 
Plate  III.,  Fig.  14.) 

Motility. — Non-motile. 

Staining  Reactions. — Stains  easily  with  the  ordinary  aniline  stains. 
The  condition  known  as  bipolar  staining  is  well-marked  in  young 
cultures.  The  Gram  method  gives  negative  results. 

Spore  Formation  absent. 

Biological  Characters. — On  Gelatine  Plates  deep  round  brownish 
colonies  appear,  while  the  surface  growth  is  limited. 

hi  Gelatine  Stab  Cultures  the  growth  resembles  a  nail  with  a  flat  top. 
(See  Photograph,  Fig.  74.) 

On  Agar-Agar  it  forms  a  thin  coating  with  crenated  margins. 

On  Potatoes  it  does  not  grow  except  they  are  rendered  alkaline,  and 
then  it  forms  a  yellowish  covering. 

Bouillon  remains  clear,  but  a  thick  sediment  is  formed. 

Milk  is  not  coagulated,  but  a  weak  acid  reaction  results.  The  indol 
reaction  is  present. 

Pathogenesis. — Rabbits,  small  birds,  and  mice  are  as  easily  affected 
as  with  chicken  cholera,  and  generally  die  in  twenty-four  hours  from 
septicaemia.  Guinea-pigs  are  not  so  easily  affected,  but  young  ones  die 
quickly.  In  all  those  animals  the  changes  at  the  point  of  inoculation 
are  more  intense  than  with  the  bacillus  of  chicken  cholera  (extensive 
haemorrhagic  oedema)  ;  fatty  degeneration  of  the  liver  is  also  frequently 
present.  Chickens  are  affected  with  large  doses.  Swine  die  from  sub- 
cutaneous injection,  with  marked  oedema  at  the  point  of  inoculation,  and 
septicaemia.  By  injection  into  the  thorax  they  die  from  a  multiple 
necrotic  pleuro-pneumonia,  the  bacilli  being  present  in  the  blood ;  there 
is  also  a  slight  enlargement  of  the  spleen,  and  catarrh  of  the  mucosa  of 
the  stomach.  Infection  by  feeding  does  not  occur.  Calves  likewise 
succumb  from  subcutaneous  inoculation  with  the  Bacillus  suisepticus. 
The  disease  essentially  consists  of  a  pleuro-pneumonia,  with  in- 
flammatory necrosis,  and  when  the  processes  become  chronic  caseous 
deposits  occur.  The  caseous  deposits  are  also  found  in  the  large  intestines 
and  adjoining  mesenteric  lymph  glands. 


Fir,.  74.— Bacillus  of  FIG.  73.— Bacillus  of 

Schwcine-seuche.      Gela-        Swine    Fever.      Gelatine 
tine  stab  culture.  stab  culture. 


Fie;.  75. — Bacillus  of  Swine 
Erysipelas.    Gelatine  stab  culture. 


Flo.  76.— Bacillus  of  Mouse 
Septicaemia.  Gelatine 
stab  culture. 


Fut.   77. — Bacillus   of   Bubonic 
Plague.  Oblique  agar  culture. 


[T.  Bon-hill,  F.K.C.V.H.,  Photo.,  Edinburgh, 


BACILLUS  OF  SWINE  ERYSIPELAS  193 

BACILLUS  OF  SWINE  ERYSIPELAS. 

(Ger.  Bac.  der  Schweine-rothlaufs ;  Fr.  Rouget  du  pore. 

Bacillus  rhusiopathise — Kitt.) 

This  disease  is  peculiar  to  the  pig,  chiefly  affecting  adult  animals, 
the  improved  breeds  being  more  predisposed.  The  characteristic 
symptoms  are  great  pyrexia,  the  appearance  of  red  or  purple 
blotches,  at  first  limited,  but  afterwards  confluent  upon  the  skin,  and 
constipation  followed  by  diarrhoea.  In  very  acute  cases  the  redness 
of  the  skin  may  not  be  present.  The  average  duration  of  the  disease 
is  about  two  days,  but  it  may  last  four  or  five  days,  and  is  sometimes 
so  severe  that  60  per  cent,  of  the  affected  animals  jiie. 

Microscopical  Appearances. — A  very  fine  cylindrical  bacillus,  1  to 
Ij  /x,  long,  and  0*2  to  0*6  /u  broad.  Morphologically  identical  with  the 
bacillus  of  mouse  septicaemia.  It  is  found  in  the  blood,  especially  in 
fine  capillaries  in  contact  with  their  internal  wall.  It  is  also  present  in 
the  exudates  in  all  the  diseased  organs,  in  the  marrow  of  the  bones,  in 
the  faecal  matters,  and  in  the  urine.  Sometimes  a  blood  corpuscle  can 
be  observed  totally  filled  with  bacilli.  (See  Photomicrograph  of  the 
allied  bacillus  of  mouse  septicaemia,  Plate  III.,  Fig.  15.) 

Motility.— Undecided  (Giinther). 

Staining  Reactions. — Stains  best  with  fuchsin,  and  beautifully  by 
the  Gram  and  Cladius  methods,  especially  in  sections  of  organs. 

Spores  are  not  formed,  but  involution  or  degenerate  forms  are  fre- 
quent. Drying  weakens  the  bacilli  quickly,  but  they  can  live  a  long 
time  in  filthy  fluids,  and  the  bacilli,  according  to  Kitt,  are  very  difficult 
to  kill  in  large  pieces  of  meat,  such  as  hams,  by  cooking,  pickling,  salting, 
or  smoking. 

Biological  Characters.  —  On  Gelatine  Plates  small  transparent, 
slightly  liquefying  colonies,  which  under  a  low  power  resemble  a  mass 
of  threads. 

In  Gelatine  Stab  Cultures  the  growth  occurs  along  the  track  of  the 
needle,  with  numerous  ramifying  out-growths  into  the  surrounding 
medium,  giving  the  growth  the  appearance  of  a  test-tube  brush.  (See 
Photograph  of  a  culture,  Fig.  75.) 

On  Agar-Agar  a  fine  coating  occurs. 

On  Potatoes  no  growth. 

In  Bouillon  slight  cloudiness,  with  the  formation  of  a  sediment. 

Indol  is  not  formed. 

Pathogenesis. — Affects  white  and  grey  mice,  white  rats,  rabbits,  and 
pigeons.  The  animals  generally  die  in  three  to  four  days,  sometimes 

N 


194  SPECIAL  BACTERIOLOGY 

later ;  the  day  before  they  die  they  sit  motionless  in  one  place,  their 
eyes  being  firmly  closed  with  secretion,  and  their  heads  retracted  as  if 
they  were  sleeping,  and  in  this  position  they  die.  Animals  can  also  be 
infected  by  feeding  with  the  virus ;  but  by  this  method  rabbits  are  less 
susceptible.  Intravenous  injection  kills  rabbits  in  three  to  six  days. 
The  field  and  wood  mouse,  guinea-pig,  ox,  horse,  ass,  dog,  cat,  chicken, 
goose,  and  duck  are  immune.  Sheep  too  seem  to  be  more  disposed 
to  infection. 

The  production  of  the  disease  in  swine  varies  according  to  the  breed ; 
well-bred  swine  die  from  subcutaneous  inoculation  by  rubbing  on  the 
skin,  and  by  feeding  with  the  virus  from  cases  of  typical  erysipelas. 

Post-Mortem  Appearances  in  Swine. — The  skin  is  oedematous, 
and  infiltrated  with  blood  ;  the  flesh  is  soft,  greasy,  and  of  a  pale  red 
colour.  The  lymph-glands,  particularly  those  of  the  mesentery,  are 
swollen  and  infiltrated  with  blood,  presenting  a  red  streaky  appearance 
due  to  engorgement  and  distension  of  the  blood-vessels  of  the  gland. 
There  is  sero-fibrinous  exudation  of  the  pleura  and  pericardium.  The 
peritoneum  is  congested  and  covered  with  ecchymosed  spots.  The 
mucous  membrane  of  the  bowel  is  highly  congested  and  swollen,  and 
in  many  places  the  epithelium  is  desquamated,  and  occasionally  partially 
formed  ulcers  are  present.  The  bacilli  are  widely  distributed,  but  not 
so  plentiful  in  the  blood  as  in  the  experimental  animals.  According 
to  Schottelius  another  bacillus  is  sometimes  found  associated  with  the 
erysipelas  bacillus  in  the  organs.  This  organism,  the  Bacillus  coprogenes 
fcetidus,  is  found  in  the  contents  of  the  bowel  and  neighbouring  organs 
of  animals  affected  with  erysipelas.  It  is  non-motile  and  shorter  than 
the  Bacillus  subtilis,  and  possesses  spores.  The  cultures  emit  a  putrid 
stench.  The  presence  of  this  organism  in  animals  affected  with  ery- 
sipelas is  of  no  etiological  importance.  Some  of  the  mild  affections  of 
swine  Urticaria  or  nettle  rash  and  skin  necrosis,  and  also  endocarditis 
verucosa  bacillosa,  are  considered  by  Kitt,  Bang,  and  Jensen  to  be 
caused  by  the  bacillus  of  swine  erysipelas.  The  faeces  of  affected  swine 
are  very  virulent.  The  disease  is  spread  by  means  of  rats  and  mice. 

Immunity. — The  repeated  passage  of  the  erysipelas  bacillus 
through  the  pigeon  renders  it  more  virulent  for  swine,  while  its 
passage  through  the  rabbit,  on  the  contrary,  diminishes  its  virulence 
for  swine.  After  a  time  the  virus  obtained  from  the  rabbit  does  not 
kill  the  swine,  but  only  makes  them  sick,  rendering  them  immune  to 
the  action  of  strong  virus.  The  degree  of  attenuation  obtained 
persists  in  cultures  made  in  ordinary  bouillon,  which  is  used  as  a 
vaccine  for  swine. 

Pasteur's  method  consists  in  using  two  vaccines  of  different  degrees 
of  virulence  in  doses  of  0*12  c.c.,  an  interval  of  ten  days  intervening 
between  the  first  and  second  inoculation.  As  young  pigs  are  not  so 


BACILLUS  OF  MOUSE  SEPTICAEMIA  195 

susceptible  to  the  disease  as  older  ones,  they  are  more  suitable  for 
immunization.  Swine  vaccinated  by  this  method  remain  immune  for 
one  year,  which  is  long  enough  for  breeding  and  fattening  purposes. 

Lorenz  has  introduced  a  method  of  protective  inoculation  with 
the  blood  serum  of  swine  previously  immunized.  The  serum  is 
obtained  in  the  following  manner : — Healthy  swine  are  first  inocu- 
lated with  serum,  and  then  with  a  virulent  culture  shortly  before 
the  blood  is  withdrawn.  Blood  serum  of  an  animal  inoculated  in 
this  manner  confers  immunity  on  other  animals.  A  pig  weighing 
eleven  stones,  supplies  on  an  average  750  c.c.  of  serum ;  out  of  this 
quantity  about  150  grammes  of  a  stable  substance  is  prepared,  con- 
taining only  i  volume  of  serum,  30  per  cent,  glycerine  and  40 
per  cent,  water.  It  is  very  difficult  to  procure  a  serum  of  uniform 
strength  in  swine  erysipelas,  because  it  is  not  easy,  and  often  impos- 
sible, to  obtain  a  uniform  grade  of  virulence  in  swine  erysipelas 
cultures.  The  serum  is  injected  subcutaneously  behind  the  ear  or 
between  the  thighs. 


BACILLUS  OF  MOUSE  SEPTICAEMIA. 

(Bacillus  Murisepticus.) 

This  is  an  experimental  infectious  disease  found  by  Koch.  The 
grey  and  white  mouse,  when  inoculated  subcutaneously  with  putrid 
blood  or  meat  infusion,  died  of  septicaemia  due  to  the  Bacillus 
murisepticus. 

Microscopical  Appearances. — Morphologically  identical  with  the 
bacillus  of  swine  erysipelas ;  the  bacilli  occur  mostly  in  the  cells.  (See 
Photomicrograph,  Plate  III.,  Fig.  15.) 

Motility.— Undecided  (Giinther). 

Staining  Reactions. — Are  similar  to  the  bacillus  of  swine  erysipelas. 

Spore  Formation. — Was  observed  by  Koch  in  one  case  (Giinther). 

Biological  Characters.  —  The  appearance  in  gelatine  stab  cul- 
tures is  very  characteristic,  the  growth  resembling  a  test-tube  brush. 
(See  Photograph,  Fig.  76).  The  growth  in  the  other  media  is  identical 
with  that  of  the  bacillus  of  swine  erysipelas. 

Pathogenesis. — Affects  grey  and  white  mice,  which  die  in  the  same 
characteristic  sitting  position  described  with  mice  inoculated  with  the 
bacillus  of  swine  erysipelas.  Field-mice,  chickens,  and  guinea-pigs  are 
immune.  Schiitz  considers  that  the  bacillus  of  mouse  septicaemia  and 
swine  erysipelas  are  probably  identical. 


196 


SPECIAL  BACTERIOLOGY 


DIFFERENTIAL  DIAGNOSIS  TABLE. 


SWINE  ERYSIPELAS. 

SWINE  FEVER. 

INFECTIOUS  PNEUMONIA 

SUISEPTICUS,             OR 
SCHWEINE-SEUCHE  . 

Size  of  the 
organism 

1  to  Belong;  0'2  to  0-6 
broad 

0-6  to  1  p  long  ;  0-4  /* 
broad 

0-6  to  1  /^  long  ;  0'4  p. 
broad 

Motility 

Non-motile 

Strongly    motile    peri- 
tricha  flagella 

Non  -motile 

Staining 

With    ordinary    stains 
and  by  the  Gram  and 
Cladius  methods 

Exhibits  polar  staining 
with    the     ordinary 
aniline  stains  ;  nega- 
tive with  the  Gram 
and  Cladius  methods 

Exhibits  polar  stain- 
ing   with    the    or- 
dinary           aniline 
stains  ;        negative 
with  the  Gram  and 
Cladius  methods 

Cultivations 

The  characteristic  test- 
tube         brush  -  like 
growth   in    gelatine 
stab    cultures  ;     no 
growth  on  potatoes 

Grows  on  agar  and  in 
gelatine     stab    cul- 
tures, nail-form  like, 
and      on      potatoes 
forms  a  yellow  coat- 
ing 

The    growth  on  agar 
and      gelatine      re- 
semble swine  fever, 
but    there     is     no 
growth  on  potatoes 
unless      they      are 
alkaline,     when     a 
yellowish      coating 
is  formed 

Indol  reaction 

None 

None 

Present 

Gas  formation 

None 

Present 

None 

Inoculation  ex- 
periments 

Mice  die  iu  two,  three, 
or  four  days,  rabbits 
in  four  to  six  days. 
Guinea  -  pigs        and 
chickens     are      im- 
mune, and  swine  over 
three  years  of  age  are 
not  affected 

Rabbits  and  mice  die 
in    seven  to  twelve 
days  ;  pigeons  with 
large      doses,      and 
chickens      are      im- 
mune.     Inoculation 
in     swine     difficult. 
Feeding  experiments 
yield    more  positive 
results 

Rabbits,  small  birds, 
and  mice  generally 
die  in  ,  twenty-four 
hours    from    septi- 
caemia,            young 
guinea-pigs     easily, 
older     ones      more 
difficult  ;     chickens 
with     large     dose. 
Feeding          experi- 
ments negative  with 
swine,       but       by 
inoculation  positive 

THE   BACILLUS   OF   BUBONIC   PLAGUE. 

The  organism  causing  this  disease  was  discovered  by  Kitasato  and 
Yersin  in  the  outbreak  of  bubonic  plague  at  Hong-Kong,  China, 
1894. 

Microscopical  Appearances.  —  Short  oval  bacilli  with  rounded 
ends,  usually  occurring  singly,  sometimes  in  pairs  and  threes,  and  very 
rarely  in  chains ;  very  often  enclosed  in  a  capsule. 

Motility . — Non-motile . 


BACILLUS  OF  BUBONIC  PLAGUE  197 

Spore  Formation. — Absent. 

Staining  Reactions. — Exhibits  polar  staining  with  the  ordinary 
staining  methods  (see  Photomicrograph,  Plate  IV.,  Fig.  20)  ;  does  not 
stain  by  the  Gram  method,  although  Kitasato  says  at  one  time  it  stains 
by  the  Gram  method,  and  at  another  time  it  is  discolorized ;  but  as 
mixed  infection  is  often  present,  the  above  peculiar  reaction  towards 
the  Gram  stain  can  perhaps  be  attributed  to  other  species  of  bacteria. 

Biological  Characters. — The  bacillus  grows  best  between  36°  and 
39°  C.,  but  also  develops  very  well  at  ordinary  room  temperature. 

On  Gelatine  Plates. — Small  darkly-defined  granular  colonies  of  a 
brownish  colour  occur,  and  the  medium  is  not  liquefied. 

In  Gelatine  Stab  Cultures. — It  develops  slowly  on  the  surface,  and 
along  the  track  of  the  needle. 

On  Agar  Plates. — In  twenty-four  hours  small  dewdrop-like  colonies 
appear,  which  in  forty-eight  hours  resemble  grey  beads  with  slightly 
iridescent  borders ;  sometimes  a  large  colony  is  observed  between  the 
smaller  ones.  The  deep  colonies  are  round  and  granular. 

On  oblique  surface  Agar  a  viscous  shiny  coating  appears ;  the  above- 
mentioned  colonies  like  dewdrops  being  sometimes  observed.  The 
water  of  condensation  is  clouded,  but  there  is  no  film  formed.  (See  Photo- 
graph, Fig.  77.) 

The  growth  on  blood  serum  is  similar  to  that  on  ordinary  agar. 

Bouillon  becomes  diffusely  clouded,  but  if  it  is  inoculated  with  a 
cohesive  mass  of  bacteria  from  an  agar  culture,  the  bacilli  develop  on 
the  bottom  of  the  tube,  while  the  upper  portions  of  the  medium  remain 
clear ;  this  mode  of  growth  being  somewhat  similar  to  that  of  strepto- 
cocci. 

Milk  is  a  bad  medium,  and  is  not  coagulated. 

Potatoes. — On  the  surface  at  37°  C.  a  scanty  greyish-white  growth 
occurs. 

The  bacillus  of  bubonic  plague  forms  no  gas  in  sugar  -  containing 
media,  and  no  indol  in  either  bouillon  or  peptone  water.  It  grows  best 
on  media  of  neutral  reaction  (Wlademiroff  and  Kressling).  The  addi- 
tion of  glycerine  to  the  media  is  detrimental. 

Vitality  of  the  Bacillus  of  Bubonic  Plague.— It  is  killed  by 
heating  ten  minutes  at  55°  C.,  and  five  minutes  at  80°  C.  Corrosive 
sublimate  1  to  1000  destroys  the  bacilli  immediately  ;  1  per  cent,  carbolic 
acid  and  1  per  cent,  lysol  in  ten  minutes.  The  mineral  acids  are  very 
effective.  Sulphuric  acid  1  to  2000  kills  the  bacilli  in  five  minutes.  Hydro- 
chloric acid  1  to  1000  in  thirty  minutes.  The  longest  time  that  infected 
material  on  lint,  wadding,  earth,  etc.,  remained  active  was  eight  days. 
Sputum  from  patients  affected  with  the  pneumonic  form  kept  in  a  vessel 
plugged  with  cotton  wool,  was  no  longer  virulent  in  sixteen  days.  In 
ordinary  drinking  water  the  bacilli  die  in  three  days,  in  sterilized  water 


198  SPECIAL  BACTERIOLOGY 

in  eight  days,  and  in  sterilized  bilge  water  in  five  days ;  also  in  direct 
sunlight  in  three  or  four  hours.— (Report  of  German  Plague  Commission.) 
The  bacilli  are  killed  by  drying  at  ordinary  room  temperature  in  four 
days. 

Pathogenesis. — The  most  susceptible  animal  is  the  rat,  a  minimum 
quantity  of  culture  being  sufficient,  when  injected  subcutaneously,  to 
cause  its  death.  The  same  results  are  obtained  when  the  virus  is  intro- 
duced on  the  mucous  membrane  of  the  nose  or  eye,  also  by  feeding 
experiments  and  by  gnawing  companions  dead  of  the  plague.  The 
latter  mode  of  infection  is  of  great  importance,  as  it  explains  the  extra- 
ordinary and  almost  inconceivable  rapidity  with  which  this  rat-pest 
spreads,  and  probably  also  it  is  the  affected  rats  that  carry  the  plague 
from  house  to  house. 

Next  to  the  rat  the  most  susceptible  animal  is  the  grey  monkey. 
All  the  ordinary  laboratory  animals  are  also  susceptible  except  the 
pigeon. 

Yersin  observed  the  bacillus  in  the  bodies  of  dead  flies  by  the 
inoculation  of  animals. 

Ogata  found  the  bacillus  in  fleas  on  rats  dead  of  the  plague. 

Nuttall  found  that  fleas  died  when  fed  with  plague  tissues  in  seven 
to  eight  days  at  12°  to  14°  C.,  and  in  about  three  days  at  23°  to  31°  C. 
In  bed-bugs  the  bacilli  soon  died. 

In  animals  subcutaneously  inoculated,  the  point  of  the  inoculation 
becomes  oedematous,  and  the  lymphatics  enlarged  in  a  few  hours ;  in 
twenty-four  hours  the  animal  is  quiet,  hair  rumpled  with  excessive 
lachrymal  discharge  from  the  eyes,  finally  convulsions  set  in,  ending  in 
death.  The  post-mortem  appearances  are  as  follows : — Bloody  redema 
at  the  point  of  inoculation,  reddening  and  swelling  of  the  lymph-glands  ; 
haemorrhagic  extravasation  into  the  abdominal  walls ;  serous  effusion 
into  the  thoracic  and  abdominal  cavities ;  intestines  congested.  Spleen  en- 
larged (see  Photomicrograph  of  section  of  spleen  of  mouse,  Fig.  78),  and 
greyish  points  resembling  miliary  tubercles  are  sometimes  present  in  the 
spleen.  The  supra-renal  bodies  are  also  enlarged.  The  characteristic 
bacilli  can  be  found  in  large  numbers  in  the  local  oedema,  lymph-glands, 
blood,  and  internal  organs. 

In  cases  where  the  animals  do  not  die  quickly,  only  local  evidence 
of  inoculation  is  present,  and  the  distribution  of  the  organism  throughout 
the  body  is  diminished. 

In  man,  when  the  virus  gains  entrance  at  the  foot,  the  superficial 
and  deep  inguinal  glands  are  the  first  to  become  affected,  and  if  the 
infection  be  through  wounds  in  the  hands,  then  the  buboes  appear  first 
in  the  axillary  region.  A  member  of  the  German  Commission  was 
infected  while  performing  an  autopsy  on  a  victim  of  plague  ;  two  days 
later  a  small  pustule  appeared  on  the  right  hand,  soon  followed  by 


r  ..-*>*  *• 

v 


1 


Fl(;>  78.*— B.  of  Bubonic  Plague.    Cover-glass  specimen  from 
spleen  of  inoculated  mouse.     Methylene  blue.     X  1000. 


Fin.  7l».—  B.  of  Broncho-Pneumonia  Bovis.     Ovoid  bacteria.     Section 
of  lung  of  American  ox.     Weigert's  method.     X  1000. 

I  am  indebted  to  Dr  G.  W.  Nuttall,  Berlin,  for  the  specimen  from  which 
this  photograph  was  taken. 


[T.  BowMll>  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


BACILLUS  OF  BUBONIC  PLAGUE  199 

lymphangitis  and  enlarged  axillary  glands.  The  plague  bacilli  were  found 
in  the  secretion  from  the  pustule.  Notwithstanding  the  very  alarming 
initial  sickness,  recovery  took  place.  In  man,  the  bacilli  are  most 
numerous  in  the  enlarged  suppurating  lymphatics.  They  are  also  pre- 
sent in  smaller  numbers  in  the  blood  and  the  internal  organs. 

Bacteriological  Diagnosis  of  Plague. — As  already  mentioned, 
the  characteristic  bacilli  are  very  numerous  in  the  pustules  or  buboes, 
and  the  polar  staining  exhibited  with  methylene  blue  enables  them  to 
be  easily  differentiated.  The  bacillus  of  chicken  cholera  resembles  it, 
but  is  somewhat  larger,  and  has  no  effect  on  man.  The  diagnosis  is  not 
so  easy  in  those  cases  where  suppuration  has  not  commenced,  and  it  is 
in  such  cases  that  it  is  most  necessary.  The  German  Commission  con- 
sider puncture  of  the  bubo  somewhat  dangerous,  on  account  of  opening 
bloodvessels,  but  the  English  doctors  make  a  long  incision  in  the 
affected  gland,  which  is  afterwards  dressed  with  antiseptics.  By  this 
method  it  is  easy  to  obtain  material  from  the  gland  for  cover-glass 
specimens,  plate,  and  other  culture  methods.  Microscopical  examina- 
tion of  the  blood  is  only  attended  with  results  in  cases  of  general 
infection.  Culture  experiments  with  the  blood  yield  better  results.  To 
separate  plague  bacilli  from  the  mixture  of  bacteria  in  sputum,  it  is  best 
to  make  gelatine  stroke  plate  cultures.  The  suspected  material  is  drawn 
across  the  surface  of  solid  gelatine,  several  strokes  being  made.  The 
plague  bacilli  grow  very  well  at  the  low  temperature  of  22°  to  25°  C., 
while  the  accompanying  bacteria,  i.e.,  diplococcus  lanceolatus  streptococ- 
cus, generally  exhibit  only  a  scanty  growth.  According  to  Hankin,  when 
the  bacilli  are  grown  on  agar  containing  2' 5  to  3" 5  of  salt  at  31°  C.,  in 
twenty-four  to  forty-eight  hours  involution  forms,  consisting  of  pear- 
shaped  bodies  and  spheres,  appear,  which  he  considers  characteristic 
enough  to  form  a  means  of  diagnosis.  Of  further  diagnostic  importance 
is  the  fact  that  the  blood  serum  of  men  and  animals  recovered  from 
plague  possesses  the  power  of  agglutination  (German  Commission).  This 
power  of  agglutination  appears  first  in  the  second  week,  and  in  the 
second  and  third  it  is  mentioned  as  being  most  pronounced.  In  the 
cases  of  mixed  infection,  particularly  those  accompanied  with  suppura- 
tion of  the  buboes,  streptococci  are  found  not  only  in  the  glands,  but 
also  in  the  blood. 

Immunity. — In  the  Pasteur  Institute  a  plague  serum  is  prepared 
from  a  highly  immunized  horse.  The  German  Commission,  experi- 
menting with  this  serum,  found  that  3  c.c.  was  sufficient  to  protect  a 
brown  monkey  against  an  after  subcutaneous  inoculation.  With  the 
susceptible  grey  monkey  10  c.c.  was  not  sufficient.  The  serum  also 
exhibited  undoubted  curative  effects  on  a  brown  monkey  inoculated 
twelve  hours  previously.  Haffkine  prepares  his  anti-plague  serum 


200  SPECIAL  BACTERIOLOGY 

from  fluid  cultures  rich  in  fat.  The  development  is  allowed  to  go  on 
for  about  a  month,  the  broth  being  shaken  now  and  then  to  ensure 
a  series  of  successive  crops.  The  organisms  are  then  killed  by 
exposure  to  70°  C.  for  an  hour.  This  is  used  as  a  vaccine,  and 
before  use  it  is  shaken  and  3  c.c.  injected.  The  efficacy  of  the  serum 
with  plague  patients  has  yet  to  be  determined. 

BRONCHO-PNEUMONIA    BOVIS. 

(Infectious  Broncho-pneumonia — Nocard  ;   Corn  Stalk  Disease — 
Billings  ;  Bacillus  Zeae — Burril.) 

The  disease  occurs  among  American  cattle  at  certain  seasons  of 
the  year,  and,  according  to  Billings,  is  caused  by  an  ovoid  belted 
organism  which  infects  cattle  eating  the  leaves  and  tender  top  shoots 
of  the  corn  stalks,  and  from  this  circumstance  he  named  it  '  The 
Corn  Stalk  Disease/  Nocard,  on  the  other  hand,  found  some  cattle 
amongst  a  lot  landed  from  America  affected  with  a  specific  lung 
disease,  which  at  the  first  glance  presented  the  appearance  of  a  recent 
lesion  of  contagious  pleuro-pneumonia ;  but  careful  microscopical 
examination  revealed  the  presence  of  ovoid  belted  organisms  that 
could  be  differentiated  in  the  lymph  spaces  resembling  a  state  of 
pure  culture.  M.  Nocard  says  :  4  This  single  character  alone  sufficed 
to  affirm  that  the  lesion  was  not  of  a  pleuro-pneumonic  kind.'  The 
author  also  discovered  the  presence  of  morphologically  identical 
organisms  in  sections  of  a  piece  of  the  lung  of  an  American  ox 
received  from  Professor  Williams  (see  Photomicrograph,  Fig.  79). 
A  portion  of  this  lung,  as  well  as  stained  sections,  were  sent  to  M. 
Nocard,  who  replied  :  '  It  is  a  bacteridian  broncho-pneumonia,  which 
in  all  probability  is  of  the  same  nature  as  that  I  have  already 
described."  In  the  absence  of  cultures,  M.  Nocard,  though  stating  his 
belief  in  the  identity  of  the  organisms,  could  not  be  absolutely 
definite. 

Microscopical  Appearances. — Short  oval  bacteria  1  //,  long,  and 
about  '5  p  in  width. 

Motility. — Strongly  motile. 

Staining  Reactions. — Stains  readily  with  alkaline  methylene  blue, 
when  the  clear  spaces  in  the  centre  of  the  organism  are  well  differ- 
entiated. By  the  Gram  method  the  reaction  is  negative.  Sections  are 
best  stained  with  methylene  blue,  according  to  Weigert's  original 
method  (see  Technique,  §  37)  ;  also  with  fuchsin. 

Biological  Characters. — According  to  Billings  the  growth  is  more 
characteristic  at  ordinary  room  temperature  than  in  the  incubator. 


PLEURO-PNEUMONIA  BOVIS  201 

On  Gelatine  Plates. — Flat,  spread  out,  bluish,  transparent,  tabulated 
colonies. 

In  Gelatine  Stab  Cultures  the  growth  takes  place  along  the  inocu- 
lation track  and  on  the  surface  of  the  medium  as  well ;  there  is  no 
liquefaction. 

Upon  oblique  surface  Gelatine  there  is  a  rapid,  pellucid,  pearly-white 
growth,  the  edges  being  scalloped. 

On  Agar-Agar  the  edges  of  the  growth  are  deeply  scalloped,  the 
separating  lines  extending  into  the  body  of  the  cultures. 

On  Potato,  greyish-white,  somewhat  elevated  colonies  develop. 

White  of  Egg. — Clear  yellow  colonies  with  slightly  raised  edges 
develop. 

Milk  is  not  coagulated. 

Pathogenesis. — Mice,  rabbits,  guinea-pigs,  and  pigeons  inocu- 
lated subcutaneously  with  2  or  3  drops  of  the  serum  or  culture  died 
in  less  than  forty-eight  hours.  Intraperitoneal  injection  caused 
death  in  fifteen  to  eighteen  hours  with  purulent  peritonitis.  Sheep 
and  calves  inoculated  subcutaneously  or  in  the  trachea  with  1  c.c.  of 
culture  serosity  or  virulent  pus  did  not  die,  but  were  seriously 
affected  for  several  days,  when  they  soon  regained  their  normal 
condition.  An  eight-month-old  calf  and  a  two-year-old  ram, 
inoculated  in  the  right  lung  with  5  drops  of  peritoneal  pus  from  a 
guinea-pig,  died  in  less  than  forty-eight  hours  with  fibrinous 
pleurisy  and  exudative  broncho-pneumonia  analogous  to  that 
observed  at  the  autopsy  on  the  American  cattle.  The  lesions 
contained  numerous  quantities  of  bacteria. — Nocard. 

Pigs,  dogs,  rats,  and  chickens  are  immune.  A  calf  and  two 
sheep  previously  inoculated  subcutaneously,  then  in  the  trachea, 
afterwards  resisted  the  effects  of  intrapulmonary  inoculation  with 
10  drops  of  a  virulent  culture. 


PLEURO-PNEUMONIA  CONTAGIOSA  BOVIS. 

Poels  and  Nolen  considered  this  bovine  scourge  was  caused  by  a 
micrococcus,  while  Arloing  isolated  a  bacillus  which  he  named  the 
Bacillus  liquefaciens  bovis,  but  it  has  been  proved  that  neither  of  these 
organisms  possess  the  pathological  significance  ascribed  to  them  by  the 
above-mentioned  investigators.  The  most  recent  announcement 
regarding  the  cause  of  this  disease  was  communicated  by  MM.  Nocard 
and  Roux,  with  the  collaboration  of  MM.  Borel,  Salimbeni,  and 
Dujardin-Beaumetz,  in  the  Revue  Veterinaire,  May  1898. 

These  investigators  placed  bouillon   previously  inseminated  with  a 


202  SPECIAL  BACTERIOLOGY 

trace  of  pleuro-pneumonic  serosity  in  sacs  of  collodion,  which  were 
carefully  sealed  and  inserted  into  the  peritoneal  cavity  of  rabbits. 
After  fifteen  to  twenty  days,  the  sacs  contain  an  opal,  slightly  turbid, 
albuminous  fluid,  but  neither  contain  cells  or  bacteria  cultivable  in 
ordinary  bouillons. 

Microscopical  Examination  of  the  contents  of  the  sacs  with  a  very 
high  power  (about  2000  diameters)  revealed  infinite,  small,  refringent, 
mobile  points  of  such  a  degree  of  fineness  that  it  is  impossible  even 
after  coloration  to  exactly  determine  their  form.  A  second  sac  con- 
taining the  identical  bouillon,  minus  the  pleuro-pneumonic  serosity,  was 
inserted  into  the  peritoneal  cavity  of  the  same  rabbit,  in  order  to 
ascertain  if  the  modifications  found  in  the  fluid  of  the  first  sac  were  not 
due  purely  and  simply  to  the  osmotic  changes  which  take  place  in  the 
vicinity  of  its  walls.  The  liquid  contained  in  this  sac  was  found  to  have 
retained  its  transparency  and  primitive  limpidity.  The  numerous 
mobile  points  that,  despite  their  extreme  fineness,  had  rendered  the 
inoculated  liquid  opalescent,  were  in  reality  living  organisms  which  had 
exhibited  infinite  growth,  in  consequence  of  the  modification  undergone 
by  the  culture  liquid,  and  thanks  to  the  obstruction  by  the  collodion  to 
the  phagocytary  action.  This  is  proved  when  two  sacs  of  inseminated 
collodion  are  inserted  into  the  peritoneum  of  a  second  rabbit — the  first 
with  a  trace  of  the  opal  liquid  thus  obtained,  the  second  with  several 
drops  of  the  liquid  previously  heated,  and  consequently  containing  no 
organisms,  comports  itself  like  the  sac  receiving  no  serosity,  and  its 
contents  remain  limpid  and  transparent,  whereas  the  other  soon 
becomes  opalescent  and  contains  the  innumerable  refringent  points 
described  above. 

Fresh  sacs  were  inseminated  with  the  opal  liquid  obtained  from  the 
fertile  sac  of  the  second  rabbit,  and  inserted  into  the  peritoneum  of  the 
third  rabbit,  and  so  on,  successively  identical  results  being  always  ob- 
tained. It  is  advisable  to  use  several  sacs  in  each  passage,  as  they  are 
frequently  ruptured.  The  rabbits  frequently  become  very  thin  previous 
to  being  destroyed,  and  sometimes  succumb  before  the  time  set  for  the 
autopsy,  when  they  are  found  in  a  profound  state  of  cachexia,  being 
nothing  more  than  skin  and  bone.  No  appreciable  organic  lesions  are 
discovered  at  the  autopsy ;  the  blood  and  pulp  of  the  parenchymatous 
organs  sown  in  various  media,  also  in  collodion  sacs,  does  not  give  rise  to 
any  cultures,  so  that  it  seems  in  all  probability  to  be  an  intoxication  due 
to  diffusion  outwards  of  the  products  elaborated  by  the  microbe  in  the 
collodion  sacs. 

It  cannot  be  attributed  in  every  case  to  digestive  or  other  troubles 
which  might  be  caused  by  the  sac  and  foreign  body,  because  several  sacs 
of  bouillon  containing  no  serosity  can  be  inserted  into  rabbits,  and 
conserved  there  for  several  months,  without  the  animals  showing  the 


FOOT  AND  MOUTH  DISEASE  203 

least  malaise,  or  losing  a  gramme  of  their  weight.  These  accidents 
appeared  more  marked,  and  the  cachexia  much  more  profound,  when  the 
sacs  introduced  after  inoculation  were  more  numerous,  of  a  greater 
capacity,  or  as  the  cultures  concerned  were  richer.  Several  attempts 
were  made  to  obtain  cultures  in  sacs  in  the  guinea-pig,  but  with  nega- 
tive results.  Even  after  remaining  six  weeks  in  the  peritoneal  cavity 
of  the  guinea-pig,  strongly  inoculated  bouillon  is  found  as  limpid  as  at 
the  commencement. 

Experimental  Inoculation. — A  small  quantity  of  the  fluid  culti- 
vated in  the  collodion  sacs  was  injected  into  five  Brittany  cows,  causing 
the  development  of  the  absolutely  characteristic  pleuro-pneumonic  en- 
gorgement ;  one  of  the  cows  succumbing  with  a  formidable  cedematous 
infiltration,  the  other  four  resisted.  Two  of  them  were  inoculated  in  the 
defended  region  with  a  strong  dose  (1  c.c.)  of  pulmonary  serosity,  and 
did  not  absolutely  manifest  any  local  or  general  symptoms,  whereas 
a  fresh  cow  inoculated  at  the  same  time  as  they  were  with  10  drops  of 
the  same  serosity  succumbed  twenty-two  days  after  inoculation.  The 
third  cow  was  re-inoculated  after  four  months  with  1  c.c.  of  pulmonary 
serosity  obtained  from  a  subacute  lesion,  but  did  not  exhibit  any  local 
lesion  or  fever.  The  fourth  cow  has  not  yet  been  re-inoculated. 


FOOT   AND   MOUTH   DISEASE. 

(Eczema  Epizootica.     Ger.  Maul  und  Klauenseuche. 
Fr.  Stomatite  Aphtheuse.) 

In  the  report  of  the  German  Commission,  published  February  1898, 
Professors  Loffler  and  Frosch  state  that  the  many  bacterial  bodies 
heretofore  described  as  the  cause  of  foot  and  mouth  disease  do  not 
possess  the  etiological  significance  attributed  to  them.  The  Commission 
were  able  to  immunize  healthy  animals  in  various  ways — calves  with 
lymph  heated  for  twelve  hours  at  37°  C.,  and  also  with  lymph  heated 
30  minutes  at  60°  C.,  and  lastly,  with  lymph  mixed  with  the  blood  of 
animals  that  had  recovered  from  the  disease  and  acquired  immunity  to 
the  introduction  of  the  virus  into  their  bodies, — the  best  results  being 
obtained  with  the  mixture  of  lymph  and  blood.  The  quantities  of  the 
blood  and  lymph  mixture  used  were  1  to  40  c.c.  of  highly  virulent  lymph  and 
10  c.c.  of  defibrinated  blood.  Some  of  the  inoculated  animals  were  not 
affected,  while  others  exhibited  slight  lesions  on  the  mouth  which  did 
not  interfere  with  their  general  health.  These  changes  appeared  ten 
to  fourteen  days  after  the  animals  were  inoculated,  as  flat,  round,  or 
rugged  exfoliations  of  the  epithelium,  and  were  localized  on  the  typical 
spots  affected  by  the  characteristic  vesicles  of  the  natural  disease. 
These  erosions  were  mostly  accompanied  by  an  infiltration  of  a  black  or 


204  SPECIAL  BACTERIOLOGY 

brownish  pigment.  The  action  is  the  same  when  the  lymph  is  injected 
into  the  vein  on  one  side  of  the  neck,  and  the  immune  blood  into  the 
vein  on  the  opposite  side  shortly  afterwards.  In  different  outbreaks  of 
the  disease  the  virulence  of  the  lymph  is  extremely  variable,  therefore 
experiments  are  necessary  to  obtain,  if  possible,  a  lymph  of  constant 
action.  Further  experiments  were  also  conducted  with  lymph  reduced 
with  thirty-nine  parts  of  water  and  mixed  with  a  considerable  quantity 
of  a  culture  of  the  Bacillus  fluorescens  (as  a  test  if  any  germs  passed 
through  the  filter),  and  the  whole  mixture  filtered  two  or  three  times 
through  a  porcelain  filter.  Cultivations  instituted  on  various  media  with 
the  filtrate  remained  sterile,  thus  proving  that  no  bacterial  elements  had 
passed  through  the  filter.  A  number  of  calves  were  injected  intra- 
venously with  this  filtrate,  and  at  the  same  time  others  with  lymph. 
The  animals  inoculated  with  the  filtrate  were  affected  at  the  same  time 
as  those  inoculated  with  the  lymph,  presenting  all  the  typical  symptoms 
of  the  disease,  high  fever,  vesicles  in  the  mouth  and  feet. 

The  Commission  explain  these  results  by  two  possibilities.  The 
bateria-free  filtered  lymph  either  contains  an  extraordinary  strong 
working  soluble  poison,  or  the  not  yet  discovered  causes  of  the  disease 
were  so  small  that  they  were  able  to  pass  through  the  pores  of  the 
filter,  which  safely  prevents  the  smallest  known  bacteria  passing  through. 

In  conclusion,  the  Commission  adopt  the  hypothesis  that  foot  and 
mouth  disease  is  caused  by  an  organism  that  is  so  small  that  it  is  able  to 
pass  through  the  porcelain  filter.  The  smallest  of  all  known  bacilli  is 
Pfeiffer's  bacillus  of  influenza,  which  is  0*5  to  1  /*  long  ;  and  if  the 
supposed  cause  of  foot  and  mouth  disease  was  only  T15  or  i  the  size  of 
those,  which  is  not  impossible,  they  would,  according  to  Professor  Abbe 
of  Jena,  be  beyond  the  limit  of  the  working  capabilities  of  our 
microscopes,  and  could  not  be  recognised  with  the  best  oil  immersion 
systems.  The  results  obtained  by  M.  Nocard  and  his  colleagues  in  their 
researches  with  pleuro-pneumonia  contagiosa  (see  page  202)  tend  to 
strengthen  the  hypothesis  advanced  by  the  Commission.  The  Com- 
mission also  consider  that  other  infectious  diseases  of  man  and  animals, 
i.e.  smallpox,  cowpox,  scarlet  fever,  measles,  spotted  typhus,  rinderpest, 
the  causes  of  which  are  as  yet  unknown,  may  possibly  belong  to  this 
group  of  extremely  small  organisms.  They  also  consider  that  the 
preparation  of  a  bacteria-free  cowpox  lymph  would  remove  the  agitation 
against  vaccination. 

CANINE    DISTEMPER. 

According  to  the  recent  researches  of  Schantyr,  three  diseases  are 
included  under  the  name  '  Distemper,'  which  can  only  be  differentiated 
from  each  other  by  bacteriological  examination. 


BACTERIA  FOUND  IN  THE  MOUTH   ,  205 

1.  Abdominal  Typhus  of  the  Dog,  caused  by  large  numbers  of  short 
bacilli  generally  found  singly  in  the  blood  and  internal  organs,  bearing 
a  great  resemblance  to  the  Bacillus  typhi  abdominalis  of  man,  especially 
in  the  growth  on  potatoes. 

2.  Dog  Typhoid,  caused  by  very  small  thin  bacilli  occurring  in  groups, 
and  staining  by  the  Gram  method. 

3.  Genuine  Canine  Distemper,  caused  by  small   slightly  curved  bacilli 
lying  in  groups,  and  not  staining  by  the   Gram  method,   growing   very 
sparingly  or  not  at  all  on  the  various  culture  media. 


BACTERIA  FOUND  IN  THE  MOUTH. 

1.  Leptothrix  Buccalis  Innominata  forms  manifold,  twisted,  and 
intertwined  motionless  threads,  which  are  stained  yellow  with  a  solution 
of  iodine  and  iodide  of  potash. 

2.  Bacillus  Buccalis  Maximus    appears   in   small   tufts   running 
parallel,  and  with  the  iodine-iodide  of  potash   solution  are  stained  blue- 
violet  (Granulose  reaction). 

3.  Leptothrix  Buccalis  Maxima   in   its   form   and   arrangement 
presents  a  great  resemblance  to  the  Bacillus   buccalis  maximus,   but  is 
stained  yellow  with  the  iodine  solution. 

4.  lodococcus  Vaginatus  is  found  in  chains  consisting  of  four  to 
ten  cells,  which  are  enclosed  in  a   sheath  which  is  about  0*75  //,  thick. 
The  cells  are  stained  a  blue-violet   with  the  iodine   solution,   while  the 
sheaths  are  stained  a  pale  yellow  colour. 

5.  Spirillum    Sputigenum    forms    comma-shaped   strongly-motile 
rods,  which,  when  two  are  arranged  together,  appear  like  the  letter  S. 

6.  Spirochaete  Dentium  (Sp.  denticola).— This  organism,  like  the 
other  above-mentioned,  is  found  under  the  edges  of  the  gums.     It  forms 
long  spirals  from  8  to  25  /z  long,  with  sharp-pointed  ends,  which  is  also 
common  to  the  spirillum  of  relapsing  fever,  whereby  both  are  distin- 
guished from  other  spirilla. 

7.  Leptothrix  Gigantica. — This  organism  was  found  in  the  coating 
of  the  teeth  of  a  dog  affected  with  Pyorrhoea  alveolaris  by  Miller,  and 
named  gigantica  on  account  of  its  immense  dimensions ;  like  the  other 
mouth   bacteria  above  mentioned,  cultivations  on  artificial  media  have 
not  yet  been  obtained. 

BACTERIA   FOUND   IN   URINE. 

Micrococcus  Urese  (Leube).  —  Cocci  0-8  to  1-0  /*  in  diameter, 
occurring  either  single,  in  pairs,  tetrads,  or  in  chains.  It  grows  on  the 
surface  of  gelatine  without  causing  any  liquefaction  of  the  medium. 


206  SPECIAL  BACTERIOLOGY 

Micrococcus  Ureae  Liquefaciens  (Flugge). — Cocci  1-25  to  2  //, 
in  diameter,  occurring  either  singly  or  in  small  chains  or  irregular 
groups.  It  liquefies  gelatine  media  slowly. 

Bacillus  Ureas  (Leube). — Forms  plump  rods  1  p  thick  with  rounded 
ends,  and  grows  on  the  surface  of  gelatine  without  liquefying  the 
medium. 

UROBACILLUS   PASTEURI. 

Found  in  putrid  urine. 

The  bacilli  are  motile  and  of  various  lengths,  and  form  threads. 
Spores  are  present,  situated  at  one  end  of  the  rod.  Urea  is  decom- 
posed by  the  action  of  the  organism. 

BACILLUS   GLISCHROGENUS. 

Found  in  slimy  urine. 

It  is  motile,  and  forms  in  urine,  milk,  and  solutions  of  starch  a  slimy 
substance. 

Pathogenesis. — When  injected  into  dogs  it  causes  nephritis  ;  in 
other  animals  pyrogenic  changes  usually  result. 


The  Chief  Bacteria  occurring  in  Air,  Soil,  and  Water. 
I.— BACILLI. 

1.  Nutrient  Gelatine  is  not  Liquefied, 
(a.)  CHROMOGENIC. 

Bacillus  Auranticus. — Small  thick  rods,  exhibiting  slight  motility. 

On  Plate  Cultures  superficial  button-shaped,  orange-coloured  colonies 
develop. 

In  Gelatine  Stab  Cultures  it  exhibits  a  shiny  orange-coloured  growth. 

In  Bouillon  the  growth  is  very  characteristic,  the  fluid  remaining 
clear,  but  a  membrane  with  isolated  orange-coloured  specks  forms  on 
the  surface,  and  a  somewhat  clear  layer  in  the  bottom  of  the  tube. 

Bacillus  Constrictus. — This  name  is  applied  owing  to  the  peculiar 
appearance  of  the  organism  when  stained  by  Zimmerman's  method. 
The  bacilli  exhibit  a  slight  constriction  between  the  individuals,  being 
united  in  short  pointed  chains  possessing  a  rod-like  appearance.  The 
colonies  on  plate  cultures  appear  as  granular  discs  with  ragged  edges, 
varying  in  colour  from  a  yellowish-grey  to  a  sulphur-yellow. 


BACILLI  207 

Bacillus  Pluorescens  Non-liquefaciens. — This  organism  occurs  as 
delicate,  short,  lively  motile  rods.  The  colonies  on  gelatine  possess  a 
glittering  appearance  like  mother-of-pearl,  and  also  exhibit  a  greenish 
fluorescence. 

On  Agar-Agar  a  greenish-coloured  growth  occurs.  Another  form  is 
described,  the  Bacillus  fluorescens  non-liquefaciens  immobilis,  which  is 
distinguished  by  its  non-motility  and  the  absence  of  flagella. 

Bacillus  Puscus. — Medium-sized  rods,  which  are  often  curved,  and 
owes  its  name  to  the  dark  brown-coloured  pigment  it  produces  in  all  the 
media. 

In  Gelatine  Stab  Cultures  the  growth  is  nail-like  at  first,  the  head 
finally  spreading  out. 

Bacillus  Rubefaciens. — Fine  rods,  united  in  two  or  more  joints. 
The  growth  in  gelatine  presents  a  pale  rose-red  colour. 

On  Potatoes  the  substratum  is  rose  coloured,  while  the  colonies  them- 
selves vary  in  colour  from  a  yellowish-grey  to  a  brownish-red. 

Bacillus  Subflavus. — This  organism  occurs  in  rods,  often  associated 
in  clusters,  and  are  two  to  four  times  as  long  as  broad.  The  cultures 
form  a  pale  yellow  pigment,  which  in  plate  cultures  shines  like  mother- 
of-pearl.  The  pigment  is  more  apparent  in  agar-agar  cultures. 

Bacillus  Brunneus. — Small  non-motile  bacillus.  In  characteristic 
cultures  the  medium  surrounding  the  growth  exhibits  a  diffuse  brown 
colour. 

(b.)  NON-CHROMOGENIC. 

Bacilli  resembling  Typhoid  Bacilli  (Weichselbaum). — This  is  a 
group  of  motile  bacilli  which  in  their  morphological  and  culture 
characteristics  resemble  the  bacillus  of  Eberth  and  Gaffky,  and  the 
Bacterium  coli  commune. 

On  Plate  Cultures  the  growth  is  similar  to  the  typhoid  and  coli 
commune  colonies. 

On  Potatoes  the  growth  is  sometimes  brown,  sometimes  yellow,  and 
sometimes  scarcely  visible. 

Milk  is  coagulated. 

Grape  Sugar  is  fermented  by  some  forms,  and  by  others  it  is  not. 

The  Nitroso  indol  reaction  is  sometimes  positive,  sometimes  negative ; 
but  the  pathogenic  effects  on  experiment  animals  is  awanting.  That 
this  group  contains  a  number  of  different  organisms  is  shown  by  the  fact 
that  it  is  not  possible  with  any  of  these  species  of  bacteria  to  produce 
immunity  against  a  second  species.  Also  in  experiments  with  the  blood 
serum  of  animals,  rendered  immune  against  one  of  these  species,  the 
power  of  causing  agglutination  in  the  culture  of  another  species  was 
always  absent. 


208  SPECIAL  BACTERIOLOGY 

2.  Nutrient  Gelatine  is  Liquefied, 
(a.)  CHROMOGENIC. 

Bacillus  Arborescens. — Slender  bacilli,  frequently  forming  wavy 
threads ;  non-motile  ;  distinguished  by  the  branch-like  ramifications  and 
iridescence  of  the  colonies  in  gelatine  plate  cultures. 

On  Potatoes  it  forms  a  yellow  to  yellowish-red  pigment. 

Bacillus  Pluorescens  Liquefaciens. — A  motile  bacillus,  very  like 
the  Bacillus  pyocyaneus.  It  liquefies  the  gelatine  very  quickly,  with  the 
formation  of  a  greenish-yellow  pigment,  brightly  fluorescent. 

On  Glycerine  Agar  the  cultures  are  quite  typical,  exhibiting  an  olive- 
green  to  olive-brown  colour. 

Bacillus  Rubidus. — Medium-sized,  strongly-motile  rods,  arranged 
in  long  threads ;  a  brownish-red  pigment  is  produced  in  gelatine  agar 
and  potatoe  cultures  ;  except  the  formation  of  pigment  it  has  scarcely 
any  other  characteristic. 

Bacillus  Violaceus. — Small,  slender,  strongly-motile  rods,  forming 
spores  in  the  middle  of  the  rods  in  agar  cultures.  Cultures  on  gelatine 
exhibit  in  the  liquid  portions  a  bluish  violet-coloured  bacterial  mass. 
On  agar  and  potatoe  media,  the  formation  of  the  pigment  is  very 
abundant,  varying  from  a  dark  violet  to  an  almost  black  colour. 

Bacillus  Viscosus. — A  bacillus  very  like  the  Bacillus  fluorescens 
liquefaciens,  but  distinguished  by  forming  a  chocolate-coloured  coating 
on  the  media. 

Bacillus  lanthinus. —  Medium-sized  motile  bacilli ;  the  appearance 
of  a  colony  growing  on  a  gelatine  plate  culture  is  compared  to  a  drop 
of  ink.  It  forms  on  all  media  a  violet  pigment. 

Bacillus  Helvolus. — Motile  rods  of  varying  length,  sometimes 
arranged  in  shorter  threads,  producing  a  yellow  to  sulphur  yellow- 
coloured  pigment.  On  plate  cultures  the  colonies  appear  as  circular 
bright  yellow  discs  lying  in  a  funnel-shaped  liquefied  cavity.  On  agar 
media  an  extensive  coating  of  an  intense  yellow  colour  is  formed. 

Bacillus  Prodigiosus. — Very  small  rods  (formerly  described  as  the 
Micrococcus  prodigiosus,  or  Monas  prodigiosa)  often  arranged  in  small 
chains,  possessing  very  slight  motility.  It  is  found  in  the  air,  less  often 
in  water,  frequently  found  in  starch  -  containing  media  (bread  and 
potatoes),  in  meat,  and  in  milk. 

It  grows  on  all  the  media,  producing  a  bright  red  colour,  which  is 
most  intense  on  potatoes,  exhibiting  a  blood-red  coloured  layer. 

On  Gelatine  Plates  the  deep  colonies  are  like  white  points,  while  the 
surface  colonies  are  round  and  red  in  colour,  with  irregular  borders. 


BACILLI  209 

The  gelatine  is  very  quickly  liquefied.  On  agar-agar  a  massive  dark 
red  substance  is  formed,  while  the  nutrient  medium  itself  is  not  coloured. 
When  cultivated  in  the  incubator  for  several  generations,  the  prodigiosus 
loses  the  red  pigment.  In  the  cultures  on  potatoes  it  also  forms,  besides 
the  red  pigment,  trimethylamin. 

Milk  is  coagulated.  Media  containing  sugar  are  fermented.  The 
prodigiosus  grows  also  in  the  absence  of  oxygen,  but  no  red  pigment 
is  produced.  It  is  somewhat  pathogenic ;  after  the  introduction  of 
large  doses  the  inoculated  animals  die  with  symptoms  of  poisoning. 

Other  chromogenic  bacteria  occur,  distinguished  only  by  the  colour 
of  the  cultures.  Bacillus  ruber  balticus,  ruber  aquatilis,  caeruleus,  pavo- 
ninus,  amethystinus. 

(b.)  NON-CHROMOGENIC. 

Bacillus  Liquefaciens. — One  of  the  most  common  and  widely 
distributed  water  bacilli.  Strongly  motile  rods,  often  arranged  in  chains 
of  four  or  more  joints.  Gelatine  media  are  liquefied  very  quickly. 

On  Plate  Cultures,  in  the  form  of  scales. 

In  Stab  Cultures,  in  the  form  of  a  stocking,  with  the  upper  portion 
distended.  The  cultures  give  off  an  unpleasant  stench.  It  is  a  faculta- 
tive anaerobe,  and  in  nutrient  media  containing  nitrates  it  produces 
nitric  acid. 

Bacillus  Liquidus. — (This  is  also  a  water  bacillus.)  Short,  plump, 
slightly  motile  bacilli,  liquefying  gelatine  very  quickly  ;  in  tubes  the 
grey  liquefied  gelatine  is  covered  with  a  thin  membrane,  which,  when 
the  tube  is  shaken,  sinks  to  the  bottom  of  the  medium. 

Bacillus  Aquatilis. — (Another  water  bacillus.)  Thin  motile  rods, 
which  liquefy  the  gelatine  slowly  (according  to  some  authorities,  not  at 
all).  In  gelatine  tube  cultures  they  grow  on  the  surface  of  the  medium 
as  small  yellow  colonies,  and  on  potatoes  with  a  scanty  yellowish  coating. 

Bacillus  Mycoides  Wurzel,  or  Root  Bacillus. — Found  in  the 
earth  and  in  certain  kinds  of  forage ;  thick,  slightly  motile  bacilli  with 
rounded  poles.  Spores  are  formed  in  the  middle  of  the  rods.  Grows 
only  in  the  presence  of  oxygen.  The  greyish-white  colonies  consist  of  a 
net  of  fine  twisted  threads.  The  gelatine  is  liquefied. 

In  Stab  Cultures  the  growth  resembles  a  pine  tree  placed  upside  down. 

On  Agar-Agar  the  growth  exhibits  a  texture  like  the  branching  of 
the  roots  of  a  tree.  There  is  another  species  called  the  Bacillus  mycoides 
roseus,  which  morphologically  resembles  the  Bacillus  anthracis ;  it  grows 
best  at  room  temperature.  On  gelatine  plates  it  forms  round,  scanty, 
quickly  liquefying,  red-coloured  colonies.  On  agar-agar  a  red-coloured 
growth ;  the  pigment  is  only  formed  in  the  dark ;  exposed  to  the  light, 
the  growth  is  white.  The  pigment  is  soluble  in  water. 

O 


210  SPECIAL  BACTERIOLOGY 

Bacillus  Subtilis  (Hay  Bacillus). — Large  motile  rods  (for  Photo- 
micrograph showing  Flagella,  see  Fig.  81),  frequently  growing  in  long 
straight  threads;  it  is  strongly  aerobic,  and  liquefies  gelatine  media 
quickly.  Optimum  temperature,  30°;  minimum,  10°;  maximum,  45°  C. 

On  Plate  Cultures,  bright  greyish-white  colonies  appear,  surrounded  by 
a  radiating  margin. 

On  Agar-Agar,  the  growth  is  peculiar,  exhibiting  a  rigid,  shrivelled, 
easily  detached  coating.  Spores  form  in  the  middle  of  the  rods,  some- 
what broader  but  considerably  shorter  than  the  mother  cell.  The  hay 
bacillus  is  found  in  the  air,  water,  dust,  faeces,  hay,  etc.  To  obtain  pure 
cultures  proceed  as  follows : — Cut  some  hay  into  small  pieces,  place  in  an 
Erlenmeyer  flask,  cover  with  water,  and  cook  for  fifteen  minutes ;  by 
this  means  all  germs  are  destroyed,  except  the  resistant  spores  of  the  hay 
bacillus.  These  grow,  and  a  thin  membrane  of  hay  bacilli  forms  in  two 
to  three  days  on  the  surface  of  the  hay  infusion.  (See  Photomicrograph, 
Fig.  80.) 

Bacillus  Mesentericus  (Potato  Bacillus). — There  are  three  forms 
of  the  organism  described  : — 

1.  B.  mesentericus  vulgatus. 

2.  B.  „  fuscus. 

3.  B.  „  ruber. 

The  last  form  which  invests  the  potato  on  which  it  is  growing  with  a 
rose  colour,  possesses  extremely  resistant  durable  forms,  which  can 
endure  boiling  for  five  to  six  hours.  The  spores  are,  in  comparison  with 
the  bacterial  cell,  very  large.  The  cultural  peculiarities  resemble  those 
of  the  hay  bacillus.  On  potatoes  the  bacillus  forms  a  strong  rugged 
coating. 

Milk  is  coagulated  and  peptonised. 

Bacillus  Spinosus.— Strongly  anaerobic  motile  rods.  In  gelatine 
it  forms  opalescent  spherical  colonies  with  bristly  outgrowths.  The 
gelatine  is  liquefied  with  the  formation  of  gas.  The  stab  culture  before 
liquefaction  resembles  a  hairy  caterpillar  (Liideritz).  The  bacillus  grows 
at  room  as  well  as  at  incubator  temperature.  The  spores  are  formed  in 
the  middle  of  the  rods,  which  become  enlarged  like  a  spindle 
(clostridium).  This  bacillus  is  found  in  garden  soil. 

II.— MICROCOCCI. 

1.  Nutrient  Gelatine  is  not  Liquefied. 

(a.)  CHROMOGENIC. 

Micrococcus  Auranticus. — Round  to  oval  cocci,  arranged  in  small 
clusters.  In  cultures  the  colonies  are  yellow,  shiny,  club-shaped,  and  do 
not  extend  very  far  over  the  media. 


FIG.  80. — B.  Subtilis  and  Spores  from  culture.     Fuchsin.     X  1000. 


Fir;.  81.— B.  Subtilis,  with  Flagelhi.     Agiir  culture.     Stained 
with  Orcciu  solution.     X  1000. 


I  7'.  Ilini'ldll,  F.R.C.V.X.,  Pkoio.,  Edinburgh,  1898. 


MICROCOCCI  211 

Micrococcus  Versicolor. — Small  cocci,  arranged  in  small  groups  or 
in  the  form  of  diplococci.  They  are  very  frequent  in  the  air.  The 
cultures  present  an  irregular  form  and  yellowish-green  colour.  On 
gelatine  they  exhibit  a  mother-of-pearl  like  iridescence,  and  cause 
fermentation  in  grape  sugar  media. 

(b.)  NON-CHROMOGENIC. 

Micrococcus  Candicans. — Round  medium-sized  cocci,  best  known 
by  their  growth  in  gelatine  stab  cultures,  in  which  they  form  a  nail-like 
growth  with  a  porcelain  white  shiny  head. 

Micrococcus  Concentricus. — Small  cocci,  arranged  like  bunches  of 
grapes  (Staphylococci),  characterised  by  the  zonary  growth  of  the 
colonies  on  gelatine  plates  and  in  stab  cultures.  The  colonies  vary  in 
colour  from  white  to  a  bluish-grey,  and  are  indented  on  the  surface. 

Micrococcus  Rosettaceus. — Medium-sized  cocci.  The  growth  is 
frequently  superficial  in  rosette-formed  masses,  with  irregular  edges  of  a 
greyish  colour,  the  central  portion  varying  from  dark  grey  to  brown. 

Micrococcus  Aquatilis. — Round  light  grey  colonies  with  a  mother- 
of-pearl  lustre,  the  edges  appearing  indented.  Under  a  low  power  the 
colonies  resemble  a  berry  in  shape. 

2.  Nutrient  Gelatine  is  Liquefied, 
(a.)  CHROMOGENIC. 

Micrococcus  Cremoides. — Small  cocci,  arranged  in  bunches,  and 
forming  a  cream-coloured  pigment.  In  the  commencement  on  gelatine 
yellowish-white  to  brownish-green  granular  circular  colonies  develop ; 
later,  the  discs  appear  fixed,  and  lie  in  a  liquefied  depression. 

Micrococcus  Agilis. — Strongly  motile  coccus,  possessing  flagella  ; 
grows  on  the  different  media,  forming  a  rose-coloured  pigment. 
Gelatine  media  are  liquefied  slowly. 

Sarcina  Lutea  (Yellow  Sarcina). — Strongly  aerobic  cocci,  arranged 
in  the  so-called  bale-like  forms.  On  gelatine  plates,  round,  slightly 
granular  yellow  colonies  develop. 

In  Stab  Cultures  a  strong  surface  growth.  The  cultures  form  a  citron 
yellow  pigment.  The  gelatine  is  liquefied  very  slowly,  and  the  citron 
yellow  growth  falls  to  the  bottom  of  the  tube,  while  the  upper  portion 
of  the  medium  remains  clear. 

Besides  the  yellow  sarcina,  there  are  white,  orange,  and  red  sarcinae, 
which  are  only  distinguished  from  the  above-mentioned  yellow  form  by 
the  different  colours  of  the  pigment  produced.  These  sarcinae  are  all 
found  in  the  air. 


212  SPECIAL  BACTERIOLOGY 


(b.)  NON-CHROMOGENIC. 

Micrococcus  Radiatus. — Small  cocci  with  no  typical  arrangement. 
On  plate  cultures  the  colonies  appear  surrounded  by  a  radiating  border. 
In  stab  cultures  the  growth  exhibits  horizontal  rays,  and  the  gelatine  is 
liquefied  slowly. 

III.— VIBRIOS. 

Vibrio  Aquatilis  (Giinther). — This  vibrio  is  distinguished  from 
the  cholera  vibrio  in  the  first  few  days  of  its  development  easily,  and 
with  certainty  by  the  character  of  its  growth.  It  forms  circular,  smooth 
edged,  finely  granular  colonies.  In  later  stages,  when  the  gelatine 
commences  to  liquefy,  a  faint  resemblance  to  the  colonies  of  the 
cholera  vibrio  appears.  The  vibrio  aquatilis  is  further  distinguished 
from  the  cholera  vibro  by  the  nitroso-indol  reaction  being  negative,  and 
the  total  absence  of  any  pathogenic  properties.  The  vibro  aquatilis  at 
first  grew  badly  in  fluid  media,  but  after  many  generations,  cultures 
were  obtained  in  bouillon  and  peptone  water.  Giinther  failed  to 
observe  the  formation  of  spirilla  with  this  vibrio. 

Vibrio  Berolinensis. — Found  by  Neisser  in  Berlin  conduit  water. 
On  gelatine  plates  the  edges  of  the  colonies  are  mostly  smooth,  and 
exhibit  a  much  more  granular  appearance  than  the  colonies  of  the 
cholera  Asiatica  vibrio ;  the  gelatine  is  liquefied  slowly,  and  the  nitroso- 
indol  reaction  is  positive.  Guinea-pigs  inoculated  intraperitoneally,  die 
with  the  same  symptoms  as  those  following  the  introduction  of  the 
genuine  comma  bacilli  of  Asiatic  cholera.  Similar  vibrios  have  been 
isolated  by  Weibel,  Loffler,  Fokker,  Kiesling,  and  also  from  the  river 
Seine. 

The  non-identity  of  this  and  the  other  vibrios  with  the  real  cause 
of  Asiatic  cholera  is  determined  by  the  negative  results  with  Pfeiffer's 
reaction  and  the  agglutination  test. 

Vibrio  Metschnikoff. — This  vibrio  was  first  found  in  an  epidemic 
amongst  chickens,  then  in  water  from  the  river  Spree.  It  is  somewhat 
thicker  and  shorter  than  the  vibrio  of  Asiatic  cholera,  often  exhibiting 
a  coccoid  formation.  It  is  stongly  motile.  The  cultures  resemble  those  of 
the  vibrio  of  Asiatic  cholera,  but  the  liquefaction  of  gelatine  media  is  more 
pronounced,  and  already  in  twenty-four  hours  there  is  a  well-marked 
nitroso-indol  reaction  present.  This  vibrio,  in  contradistinction  to  Koch's 
vibrio,  is  just  as  pathogenic  for  pigeons  as  for  guinea-pigs.  (See  Photo- 
micrograph from  blood  of  inoculated  pigeon,  Fig.  82.) 

Vibrio  Gindha  (Pasquale). — Found  in  well  water  at  Massauah ; 
somewhat  long,  slightly  bent  rods,  stongly  motile,  possessing  one  fla- 
gellum.  Slightly  pathogenic  ;  nitroso-indol  reaction  negative. 


vsr 


'lo.  82.— V.  Metschnikoff,  from  blood  of  inoculated  pigeon 
Fuchsin.     X  1000. 


Fu;.  S2t(.—  Troiuinelscliliiger  Bacilli.    Cover-glass  specimen  from 
agar  culture.    Fuchsin.     X  1000. 


[7".  Una-hill,  F.R.C.V.S.,  Photo.,  Edinburgh,  18»8. 


BACTERIA  FOUND  IN  MILK  213 

Vibrio  Lissalbon. — Obtained  in  a  widespread  cholera  epidemic  in 
Lisbon,  in  which  only  one  death  occurred.  On  gelatine  plates  it  forms 
circular,  sharply  limited,  slightly  liquefying,  whitish-yellow  colonies. 
Nitroso-indol  reaction  negative. 

Vibrio  Phosphorescens  (Dunbar). — Isolated  from  the  river  Elbe. 
Morphologically  and  in  cultures  it  resembles  the  vibrio  of  Asiatic  cholera, 
but  is  distinguished  by  being  phosphorescent. 

Vibrio  Massauah. — This  vibrio  possesses  two  to  four  flagella,  while 
the  vibrio  of  Asiatic  cholera  has  only  one.  The  nitroso-indol  reaction  is 
positive.  It  is  pathogenic  for  pigeons,  guinea-pigs,  and  rabbits. 

BACTERIA  FOUND   IN   MILK. 

Bacteria  are  always  present  in  milk  unless  it  is  drawn  from  the 
udder  under  sterile  precautions.  Milk  can  be  contaminated  in  various 
ways :  from  the  gland  direct,  i.e.,  B.  tuberculosis,  etc. ;  c  by  the 
hands  of  the  milker,1  dirty  vessels,  hay  dust,  etc.,  and  by  the  water 
added  by  the  thrifty  dairyman.  Milk  is  an  excellent  medium  for  the 
development  of  many  forms  of  bacteria,  and  under  favourable  condi- 
tions, temperature,  etc.,  it  quickly  undergoes  changes. 

Pathogenic  bacteria  may  also  contaminate  milk.  The  organism 
most  frequently  found  being  the  B.  tuberculosis.  Pyogenic  cocci  have 
also  been  found.  From  contamination  with  diseased  matter  from 
affected  persons  and  premises,  milk  may  be  the  means  of  carrying  and 
spreading  typhoid  fever  and  diphtheria  (and  according  to  some 
authorities,  scarlet  fever).  Such  organisms  are,  however,  easily 
destroyed  ;  the  most  resistant  of  all,  the  B.  tuberculosis,  being  killed 
by  Pasteurizing  the  milk  for  thirty  minutes  at  70°  C.,  or  by  cooking 
the  milk  for  ten  minutes,  whereby  it  is  heated  between  90°  and  95°  C. 
Nothing  now  remains  after  the  process  of  sterilization  except  the 
resistant  spores  of  some  bacteria.  (For  special  methods  of  examining 
and  staining  bacteria  in  milk,  see  Technique,  §  20.) 

A.— COCCI. 
MICROCOCCUS   ACIDI   LACTICI. 

Found  in  fresh  milk,  occurring  either  as  single  large  cocci  or 
diplococci. 

Biological  Characters. — Aerobic. 

On  Gelatine  Plates. — Forms  small  yellowish  non-liquefying  colonies. 
Milk  is  first  coloured    red,  then    coagulated,   finally   becoming   de- 
colorized. 


214  SPECIAL  BACTERIOLOGY 

SPH^ROCOCCUS   ACIDI  LACTICI. 

Found    in    fresh    milk;    small    cocci,    occurring   as  diplococci   or 
arranged  in  clusters. 

Biological  Characters. — Facultative  anaerobe. 

On  Gelatine  Plates. — Round,  white,,  non-liquefying  colonies. 

Milk  is  coloured  red  and  coagulated  with  the  formation  of  an  acid. 

STREPTOCOCCUS   ACIDI   LACTICI. 

Found  in  curdled  milk ;  small  cocci,  arranged  in  chains. 

Biological  Characters.— 

On  Gelatine  Plates. — Round,  white,  non-liquefying  colonies. 

In  Gelatine  Stab  Cultures  the  growth  is  entirely  confined  to  the  stab. 

Milk  is  coagulated  with  the  formation  of  an  acid. 

MICROCOCCUS   ACIDI   LACTIS   LIQUEFACIENS. 

Found  in  butter ;  oval  cocci,  occurring  in  diplococci  or  tetrads. 

Biological  Characters. — Facultative  anaerobe  ;  optimum  tempera- 
ture 21°  C. 

On  Gelatine  Plates. — Small  round,  white,  liquefying  colonies. 

In  Gelatine  Stab  Cultures. — A  liquefying,  funnel-shaped  growth,  with  a 
film  on  the  surface. 

Milk  is  coagulated  with  the  formation  of  an  acid.  The  casein  is  not 
peptonised ;  in  one  to  two  weeks  it  acquires  a  musty  odour. 

STREPTOCOCCUS  HOLLANDICUS. 

Found  in  the  ropy  whey  used  in  making  Edam  cheese. 

Microscopical  Appearances. — Occurs  in  pairs  and  frequently  in 
long  chains. 

Actions. — When  sterile  milk  is  inoculated  with  this  organism  it 
becomes  sour  and  ropy  in  twelve  to  fifteen  hours  at  25°  C. 


B.  -BACILLI. 
BACILLUS   ANAEROBIUS   (FLUGGE),   II.,   III.,   IV. 

Three  different  species  of  rods  found  several  times  by  Flugge  in 
milk  that  was  cooked  one  and  a  half  hours.  Species  III.  and  IV. 
form  spores. 


BACILLUS  CYANOGENUS  215 

Biological  Characters. — They  are  all  anaerobic  and  liquefy  gelatine 
media  very  quickly,  and  form  gas  quickly  in  sugar-containing  media. 
In  Milk,  No.  II.  causes  coagulation  without  putrid  gas  formation. 
No.  III.  has  no  effect. 
No.  IV.  coagulates  milk  with  putrid  gas  formation. 

Pathogenesis. — Nos.  III.  and  IV.  are  poisonous  to  animals,  while 
No.  II.  is  not. 


BACILLUS   CYANOGENUS   (Bacillus  of  blue  milk). 

This  organism,  the  cause  of  blue-coloured  milk,  was  first  cultivated 
by  Hueppe  on  gelatine  plates. 

Microscopical  Appearances.  --  Bacilli  of  various  dimensions 
according  to  Hueppe  and  Flugge,  0*3  to  0'5  by  1  to  4  p. 

Motility. — Motile,  the  flagella  being  arranged  in  a  bundle  at  one 
end  (Lophotricha). 

Spore  Formation. — Absent.  The  spores  described  by  Hueppe  are 
considered  by  Heim  as  nothing  but  involution  changes. 

Staining  Reactions. — Stains  with  the  ordinary  dyes,  but  not  by 
the  Gram  method. 

Biological  Characters. — Grows  best  at  ordinary  room  temperature ; 
at  37°  almost  no  growth  takes  place. 

On  Gelatine  Plates. — Greyish-white,  granular,  non-liquefying  colonies 
with  scalloped  edges. 

In  Stab  Cultures  the  deep  growth  is  very  limited. 

On  A  gar. — Greyish-blue  growth. 

On  Potatoes. — A  yellowish  shiny  coating.  Two  pigments  are  formed, 
a  blue  and  a  fluorescent  colour.  In  gelatine  cultures  the  fluorescence 
appears  first,  and  later  the  blue-black  pigment.  The  latter  develops 
more  luxuriantly  on  agar  media. 

In  Milk  the  blue  pigment  is  only  formed  in  the  presence  of  an  acid. 
In  non-sterile  milk  blue  spots  appear  at  first  on  the  cream ;  finally  the 
whole  surface  is  coloured  a  sky  blue. 

In  Sterile  Milk  a  grey  colour  develops,  which  only  turns  blue  on  the 
addition  of  an  acid.  If  grape  sugar  is  added  to  sterile  milk  it  is  coloured 
blue  by  the  bacillus,  an  acid  being  formed  from  the  sugar. 

Pathogenesis. — Non-pathogenic. 

This  bacillus  was  supposed  to  cause  the  blue  colour  in  cheese,  but 
such  a  supposition  is  untenable  in  view  of  the  inoculation  experiments 
conducted  by  Adametz  and  Beyerinck. 


216  SPECIAL  BACTERIOLOGY 

BACILLUS  ACIDI  LACTICI  (HUEPPE). 

Found  in  sour  milk.  This  organism  is  without  doubt  identical 
with  the  Bacterium  lactis  discovered  by  Lister  in  1877.  Lister  also 
discovered  that  lactic  acid  bacteria,  although  frequently  found  in 
dairies,  are  very  seldom  found  in  the  open  air. 

Microscopical  Appearances. — Non-motile  rods  1-0  to  1-7  /x  long, 
and  0'3  to  0'4  /x  broad,  mostly  in  pairs,  but  sometimes  arranged  in 
small  chains. 

Spore  Formation  absent. 

Biological  Characters. — Facultative  anaerobe  ;  optimum  tempera- 
ture, 37°  C. 

On  Gelatine  Plates. — Flat  diffuse  superficial  colonies  with  irregular 
borders,  resembling  those  of  Bacterium  coli  commune. 

In  Gelatine  Stab  Cultures. — A  nail-shaped  growth. 

On  Agar. — A  yellowish-white  coating. 

On  Potatoes. — A  yellowish-brown  coating. 

In  Milk,  it  forms  an  acid  which  precipitates  the  casein,  producing 
alcohol  and  CO2. 

The  above  organism  is  not  the  only  organism  that  changes  milk- 
sugar  into  lactic  acid,  but  it  is  probably  the  common  cause  of  the 
spontaneous  souring  of  milk. 

BACILLUS  LACTIS  ACIDI  (LEICHMANN). 

This  bacillus  was  found  by  Leichmann  in  milk,  which,  when 
maintained  at  50°  C.,  underwent  spontaneous  lactic  acid  fermentation. 
When  cultivated  in  sterile  milk  it  forms  lactic  acid,  which  turns 
polarized  light  to  the  left. 

BACILLUS    LACTICUS   (GUNTHER  AND  THIERFELDER). 

Found  in  sour  milk. 

Non-motile  bacilli,  0*5  to  0*6  by  I/O  /x,  usually  arranged  in  pairs 
or  small  chains.  It  does  not  produce  spores,  but  stains  by  the  Gram 
method. 

Biological  Characters. — Grows  best  at  28°  C.,  and  better  on  media 
containing  sugar  than  on  the  ordinary  media. 

Gelatine  Media  are  not  liquefied.  The  developing  colonies  remain 
very  small,  but  well  marked. 

On  Potatoes  the  development  is  very  scanty. 

In  Bouillon. — A  turbid  growth  which  does  not  cause  any  change  in 
the  reaction. 


BACILLUS  LACTIS  217 

In  Grape  and  Milk  Sugar  Bouillon  the  growth  is  luxuriant,  the  media 
becoming  strongly  acid  without  the  production  of  gas. 

To  obtain  pure  culture  of  the  organism  from  sour  milk  proceed  as 
follows : — 

1 .  Melt  three  tubes  of  gelatine  in  the  water-bath  at  30°  C. 

2.  Put  some  CaCO3  in  a  clean  tube,  add  a  little  water,  sterilize  in 
the  steamer,  or  boil  over  the  Bunsen  flame. 

3.  Inoculate  the  first  or  original  tubes  with  three  platinum  loops  of 
milk,  and  reduce  in  the  usual  manner,  using  five  platinum  loops  of  milk. 
Mix  the  milk  and  gelatine  thoroughly. 

4.  Put  a  few  drops  of  the  solution  of  CaCO3  in  three  sterile  Petri- 
dishes,  pour  the  inoculated  gelatine  on  the  CaCO3,  and  see  that  they 
are  thoroughly  commingled. 

After  development  each  acid-forming  colony  will  be  found  surrounded 
by  a  transparent  field. 

BACILLUS  LACTIS  (FLUGGE). 

Found  in  bitter  milk.  Flugge  isolated  eleven  different  varieties, 
which  all  belong  to  the  group  of  hay  bacilli.  They  possess  the  power 
of  peptonising  the  casein  of  the  milk,  whereby  it  acquires  a  bitter  taste. 
A  few  of  the  varieties  produce  toxic  substances  which,  when  given  to 
young  dogs  per  os,  caused  diarrhoea,  muscular  weakness,  and  falling  of 
the  temperature.  They  form  very  resistant  spores,  which  are  not 
destroyed  by  several  hours'  cooking.  It  is  on  account  of  these  spores 
that  the  sterilization  of  milk  is  so  difficult.  If  improperly  sterilized  milk 
is  placed  at  a  high  temperature,  the  spores  germinate,  the  above- 
mentioned  poisonous  substances  being  formed.  Some  authors  consider 
that  these  substances  may  be  the  cause  of  summer  diarrhoea  in  children. 

The  morphological  characters  of  these  eleven  species  of  bacteria  are 
similar  to  those  of  the  Bacillus  subtilis  (see  page  210).  They  are  all  motile 
rods  of  various  lengths,  and  all  liquefy  gelatine  media.  (See  also  Bacillus 
Anaerobius  (Flugge),  2,  3,  and  4,  page  214). 

BACILLUS  LACTIS  INOCUUS. 

Found  in  the  faeces  of  infants  and  in  milk  ;  short,  non-motile 
rods.  In  the  animal  body  it  forms  capsules. 

Biological  Characters. — Aerobic. 

On  Gelatine  Plates  white,  round,  non-liquefying  colonies  develop. 

On  Potatoes  a  brownish  coating. 

Milk  is  not  changed.  In  grape  sugar  agar  no  gas  is  formed.  Indol 
is  not  formed. 

Pathogenic  only  in  very  large  doses. 


218  SPECIAL  BACTERIOLOGY 


BACILLUS  LACTIS  ALBUS  (LOFFLER). 

Found  in  butter-milk  ;  very  long  motile  bacilli,  arranged  sometimes 
in  threads.  Spore  formation  present. 

Biological  Characters. — Gelatine  media  are  liquefied. 

On  Agar  a  thick  whitish  coating  develops. 

On  Potatoes  dry  white  colonies. 

Milk  is  coagulated,  and  the  casein  peptonised. 

BACILLUS  LACTIS  (BLEISCHII). 

Found  in  butter-milk ;  large  motile  bacilli,  forming  spores. 
Biological  Characters. — Facultative  anaerobe. 
Gelatine  Media  are  liquefied. 

On  Agar  and  Potatoes  a  light  grey  coating.  The  spores  are  very 
resistant,  and  are  only  killed  after  being  cooked  for  six  hours. 

BACILLUS  LACTIS  ERYTHROGENES  (HUEPPE). 

Found  in  red-coloured  milk  ;  short,  non-motile  rods  0'3  to  0'5  by  1  to 

1-4   fJL. 

Spore  Formation  absent. 

Biological  Characters. — On  Gelatine  Plates,  round,  yellow,  gradually 
liquefying  colonies ;  the  gelatine  surrounding  the  colonies  is  coloured 
red. 

In  Gelatine  Stab  Cultures  the  development  is  very  slow ;  and  kept  in 
a  dark  place,  the  medium  is  coloured  red. 

On  Agar  and  Potatoes  a  yellowish  coating  develops,  while  the  sur- 
rounding medium  is  coloured  a  faint  red. 

Milk  is  fermented  and  peptonised,  obnoxious-smelling  gases  being 
formed  ;  it  is  at  first  a  dirty  red,  then  brownish-red,  and  finally  blood- 
red  coloured.  The  yellow  pigment  in  the  colonies  is  soluble  in  all 
extractive  materials,  while  the  red  pigment  is  also  soluble  in  water. 

Pathogenesis.  — The  red  milk  is  not  pathogenic  for  man. 

BACILLUS  LACTIS  PITUITOSI  (LOFFLER). 

Obtained  by  Loffler  from  milk. 

Microscopical  Appearances. — Somewhat  thick,  slightly  bent  rods, 
that  soon  break  up  into  coccoid-like  segments. 

Biological  Characters. — In  gelatine  it  forms  white  colonies  which, 
by  transmitted  light,  appear  brown-coloured,  usually  sharply  outlined, 
but  sometimes  indented. 

On  Agar,  dirty  white-like  colonies  develop. 


BACTERIA  CAUSING  ROPINESS  IN  MILK  219 

On  Potatoes  a  greyish-white,  pearl-like,  somewhat  dry  coating. 

Milk  becomes  slightly  acid  and  slimy,  giving  off  a  quite  specific 
odour. 

Whether  this  ropy  mass  is  formed  from  the  milk  sugar  or  from  the 
casein  has  not  been  determined. 

BACILLUS  LIMBATUS  ACIDI  LACTICI. 

Found  in  milk. 

Microscopical  Appearances.  —  Short,  non-motile  rods,  mostly 
arranged  in  diplococci.  Capsules  present.  Spore  formation  absent. 

Biological  Characters. — On  Gelatine  Plates  round,  white,  non-lique- 
fying colonies. 

In  Gelatine  Stab  Cultures  the  growth  is  mostly  on  the  surface. 

Milk  is  coagulated  a  red  colour,  an  acid  being  developed. 

BACTERIUM  ACIDI  LACTICI  (GROTENFELD). 

Found  in  sour  milk.  Small,  non-motile  bacteria  O3  to  0*4  by  1  to 
1-4  p. 

Biological  Characters. — Facultative  anaerobe ;  optimum  tempera- 
ture, 37°  C. 

On  Gelatine  Plates  round  porcelain-white  colonies. 

In  Gelatine  Stab  Cultures  a  nail-like  growth. 

On  Potatoes  a  greyish  covering. 

Bacteria  causing  Ropiness  in  Milk. 
I.— BACILLUS  GUMOSUS. 

Found  in  slimy  milk  ;  large,  slightly  motile  bacteria. 
Spore  Formation  present. 

Biological  Characters. — Gelatine  medium  is  liquefied  slowly. 
On  Agar  and  Potatoes  a  puckered  white  coating.     Cane  sugar  is  fer- 
mented, and  when  alcohol  is  added  to  the  solution  gum  is  formed. 

II.— BACILLUS  VISCOSUS  LACTIS  (ADAMETZ). 

Found  in  water. 

Microscopical  Appearances. — Non-motile  bacilli,  1-1  to  1-3  by 
1 '2  to  1*7  /*;  sometimes  occurring  in  threads  with  capsules. 

Biological  Characters. — In  Gelatine  Media  the  deep  colonies  are 
small,  while  the  surface  colonies  exhibit  an  extensive  development  of 
slimy  drops  with  serrated  edges. 


220  SPECIAL  BACTERIOLOGY 

On  Agar  a  dirty  white,  ropy,  slimy  coating  develops. 
Milk  at  ordinary  temperature  in  five  to  ten  days  becomes  ropy,  and 
finally  transparent,  from  disintegration  of  the  milk  globules. 

BACTERIUM  ACIDI  LACTICI  (PETERS). 

Found  in  sour  dough.     Short  motile  rods,  0'4  to  1'5  p. 

Biological  Characters. — On  Gelatine  Plates  round  colonies  with 
concentric  stratification  of  a  light  red  colour.  In  solutions  of  sugar,  to 
which  some  yeast  is  added,  this  organism  produces  great  quantities  of 
lactic  acid. 

Bacteria  causing   Acetic  Acid   Fermentation. 
BACILLUS  ACETICUS  (HANSEN). 

(Mycoderma  Aceti.) 

The  Mycoderma  aceti  was  considered  by  Pasteur  to  be  the  cause 
of  the  acetic  acid  fermentation  in  wine  and  beer.  (Hansen  divides  it 
into  two  forms,  Bacillus  aceticus  and  Pasteurianus.) 

Microscopical  Appearance. — Short,  non-motile  bacilli,  forming 
threads  and  frequently  involution  forms. 

Spore  Formation  absent. 

Staining  Reactions. — Stains  by  the  Gram  method. 

Biological  Characters. — Aerobic ;  optimum  temperature,  30°  to 
40°  C. 

On  Solid  Media  it  grows  on  the  surface,  forming  porcelain  cup- 
like  colonies,  especially  on  Beer  Gelatine  ;  prepared  by  adding  5  per  cent, 
gelatine  to  beer. 

In  Liquid  Media  a  membrane  forms  on  the  surface,  the  underlying 
liquid  being  slightly  clouded. 

Specific  Actions. — It  oxidises  alcohol,  forming  acetic  acid,  and  further 
splitting  up  the  latter  into  CO2  and  H2O.  It  is  distinguished  from  other 
members  of  the  group  by  staining  yellow  when  treated  with  a  solution 
of  iodine. 

BACILLUS   PASTEURIANUS  (HANSEN). 

(Mycoderma  Pasteurianum). 

This  bacillus  is  found  in  beer  and  wine,  but  does  not  occur  as 
frequently  as  the  previous  organism,  from  which  it  is  distinguished  by 
giving  a  blue  reaction  with  solution  of  iodine. 


BACTERIA  CAUSING  FERMENTATION  221 


BACILLUS    ACETICUS    PETERSII. 

Found  in  old  sour  dough ;  resembles  the  Bacillus  aceticus  of  Hansen. 
Forms  threads  ;  is  strongly  aerobic  ;  on  gelatine  slimy  colonies  develop. 


Bacteria   causing   Butyric   Acid   Fermentation. 
BACILLUS  BUTYRICUS   (BOTKIN). 

Found  in  water,  milk,  and  manured  earth. 

Microscopical  Appearances. — Long  motile  bacilli,  forming  threads. 

Spore  Formation  present,  situated  in  the  middle  of  the  rods,  very 
resistant,  and  are  not  killed  during  the  process  of  sterilizing  milk.  As 
they  do  not  germinate  under  18°  C.,  milk  sterilized  for  children  should 
be  kept  at  a  lower  temperature  before  use. 

Staining  Reactions. — Stains  by  the  Gram  method. 

Biological  Characters. — Anaerobic. 

On  Gelatine.  Plates. — Round  or  oval  liquefying  colonies,  developing  an 
odourless  gas. 

In  Milk  at  the  bottom  of  the  culture  a  layer  of  serum  forms,  out  of 
which  the  gas  rises  upwards ;  the  milk  is  coagulated.  The  coagulated 
albumen  rises  to  the  surface  and  is  peptonised,  so  that  eventually  only 
the  fat  swims  on  the  surface.  Butyric  and  allied  fatty  acids  are  formed. 
In  media  containing  sugar  involution  forms  of  the  bacilli  occur. 

BACILLUS  BUTYRICUS  (PRAZMOSKI). 

(Clostridium  Butyricum.) 

Found  in  putrid  vegetable  infusions. 

Microscopical  Appearances. — Strongly  motile  bacilli,  about  1  //. 
broad,  of  various  length,  forming  threads. 

Spore  Formation  present,  situated  in  the  middle  of  the  rod, 
causing  a  spindle-formed  swelling.  The  spores  are  1  //,  broad  and  2  to 
2'5  fjb  long.  When  the  mature  spore  germinates  the  germinating  rod 
escapes  at  one  end,  while  the  spore  membrane  remains  attached  at  the 
other  parts  of  the  young  bacillus  like  a  cap. 

Staining  Reactions. — With  a  watery  solution  of  iodine  the  bacilli, 
when  cultivated  on  media  containing  starch,  are  stained  blue,  whereby  it 
is  called  '  The  Bacillus  Amylobacter' 


222  SPECIAL  BACTERIOLOGY 

Biological  Characters. — Strongly  anaerobic.  In  solutions  of  starch, 
dextrin,  sugar,  and  lactic  acid  salts  a  great  quantity  of  butyric  acid  is 
formed,  accompanied  by  the  development  of  H  and  CO2. 

BACILLUS   BUTYRICUS   (HUEPPE). 

Microscopical  Appearances. — Large  rods,  frequently  occurring  in 
pairs. 

Spore  Formation  present,  situated  in  the  middle  of  the  rods. 

Biological  Characters. — Grows  in  the  presence  of  oxygen  on  the 
ordinary  media  at  both  room  and  incubator  temperature. 

Gelatine  Media  are  quickly  liquefied. 

On  the  surface  of  A  gar  Media,  a  moist  yellowish  coating. 

In  Sterile  Milk  it  develops  best  at  incubator  temperature,  when 
coagulation  occurs  similar  to  that  produced  by  rennet,  without  the 
reaction  of  the  milk  being  changed.  The  casein  is  next  dissolved  and 
changed  into  peptone  and  other  products,  ammonia  being  produced. 
At  the  same  time  the  milk  acquires  a  bitter  taste.  This  bacillus  forms 
butyric  acid  out  of  lactic  acid  salts. 

Bacteria  causing  Specific  Changes  in  Beer,  Wine,  and  Sugar. 
BACILLUS   VISCOSUS   CEREVISLE   (VAN   LAER). 

Found  in  ropy  beer,  yeast,  in  the  air,  and  on  slimy  bread. 

Microscopical  Appearances. — Rods  0-8  to  1-6  by  2  to  4  /z,  seldom 
occurring  in  chains  ;  said  to  form  spores  situated  in  the  end  of  the 
rods. 

Biological  Characters. — In  Gelatine  Stab  Cultures  the  growth  is 
uniform  along  the  inoculation  track,  and  on  the  surface  a  diffuse  white 
growth  develops.  The  colonies  are  sharply  circumscribed,  and  when 
examined  under  a  low  power  appear  brown  coloured ;  old  colonies  are 
serrated,  and  in  the  middle,  white,  curly,  and  slightly  thready. 

In  Beerwort  at  27°  C.  a  germine  ropiness  occurs  in  twenty-four  hours, 
a  large  amount  of  CO2  being  formed  ;  the  surface  being  studded  later 
with  yellowish,  ropy,  slimy  islets. 

Milk  and  Solutions  of  Peptone  and  Cane  Sugar  also  become  ropy,  gas 
being  formed. 

On  Potatoes,  white,  warty,  viscous  colonies  develop,  giving  off  a  smell 
like  decayed  fish.  (Van  Laer  also  describes  another  similar  organism, 
which  is  distinguished  from  the  above  by  only  producing  slight 
fermentation  and  ropiness.)  The  injurious  influence  of  this  organism  is 
only  manifest  when  it  obtains  access  to  the  wort  before  the  primary 


BACTERIA  CAUSING  SPECIFIC  CHANGES  223 

fermentation.  When  added  afterwards  no  injurious  actions  were 
observed.  The  ropiness  in  the  beer  is  due  to  two  mucilaginous 
substances  produced  by  this  organism ;  one  contains  nitrogen  and  is 
insoluble,  while  the  other  contains  no  nitrogen  and  is  soluble  in  water. 

BACILLUS    VISCOSUS   SACCHARI   (KRAMER). 

Found  in  slimy  solutions  of  sugar. 

Microscopical  Appearances. — Small,  non-motile  rods,  forming 
threads  but  no  spores. 

Biological  Characters. — Optimum  temperature,  22°  C. 

Stab  Cultures  in  cane  sugar  gelatine  liquefy  quickly,  an  adherent 
sediment  being  deposited. 

Sugar  solutions  containing  besides  the  necessary  nitrogenous  sub- 
stances for  the  growth  of  the  bacteria,  are  changed  into  slime. 

BACILLUS   VISCOSUS   VINI   (KRUMER). 

Found  in  slimy  wine. 

Microscopical  Appearances.  —  Non-motile  bacteria  of  various 
lengths,  forming  threads. 

Biological  Characters. —  Strongly  anaerobic  ;  optimum  tempera- 
ture, 18°  C.  Develops  only  on  wine  and -glucose  solutions.  Wine  is 
fermented  in  one  to  two  months,  a  thick  slime  being  formed. 

'      LEUCONOSTOC  MESENTEROIDES  (CIENKOWSKI). 

(Froschlaich  pilz.     Pilz  der  Dextrangarimg.     Frog  spawn  fungus.) 

Found  on  beetroot  juice  and  molasses  of  sugar  factories,  where  it 
develops  in  large  gelatinous  masses,  resembling  frog  spawn.  It  is 
also  found  on  raw  or  cooked  carrots  and  sugar  beets. 

Microscopical  Appearances. — It  forms  chains  of  spherical  or  oval 
cocci  from  1*8  to  2  p  in  diameter,  enclosed  within  a  thick,  tough, 
membranous  envelope.  Finally,  owing  to  the  anastomoses  of  numerous 
chains  they  appear  as  large,  compact,  gelatinous,  zooglceic  masses. 

Staining  Reactions. — The  cover-glass  specimen  is  first  stained  with 
dahlia  violet,  which  stains  the  cocci,  and  then  immersed  in  an  aqueous 
solution  of  rosolic  acid,  which  stains  the  gelatinous  envelope  a  rose-red 
colour. 

Biological  Characters. — It  is  a  facultative  anaerobe ;  optimum 
temperature,  30°  to  37°  C.  It  is  very  difficult  to  obtain  in  pure  cultures, 


224  SPECIAL  BACTERIOLOGY 

owing  to  the  gelatinous  mass  being  contaminated  with  various  fungi ;  to 
overcome  this  difficulty  heat  the  cultures  continuously  for  fifteen 
minutes  at  75°  C.,  in  order  to  destroy  the  fungi.  The  gelatinous 
envelopes  only  develop  in  cultures  on  cane  or  grape  sugar  media,  and  in 
a  short  time  the  growth  acquires  great  dimensions.  Scheibler  considers 
the  gelatinous  substance  to  be  dextrin. 

On  ordinary  Gelatine  the  growth  exhibits  no  special  characteristics. 
On  Gelatine  containing  grape  sugar  it  is  very  diagnostic ;  consisting  in 
ten  to  fourteen  days  of  a  whitish  confluent  mass,  with  slimy,  hyaline, 
gelatinous  lumps  on  the  surface.  During  the  first  eight  days  the  growth 
exhibits  a  dry  elastic  consistence,  but  during  the  next  few  weeks  it 
becomes  softer,  moister,  finally  forming  a  soft  pulp.  Individual  colonies 
resemble  wart-like  balls,  sometimes  spread  out  with  a  puckered  film  on 
the  surface. 

In  Grape  Sugar  Gelatine  Stab  Cultures  proliferations  of  various 
dimensions  occur  along  the  inoculation  track. 

As  already  mentioned,  the  gelatinous  substance  is  only  formed  in 
material  containing  grape  or  cane  sugar.  Other  carbohydrates  tested  by 
Leisenberg  and  Zopf  were  found  unsuitable.  The  organism  produces 
Invertin,  which  splits  up  the  cane  sugar.  It  ferments  lactose,  maltose, 
and  dextrin,  forming  lactic  acid  with  a  faint  evolution  of  gas.  The 
addition  of  3  to  5  per  cent,  of  calcuim  chloride  to  the  nutrient  medium 
favours  the  production  of  mucus  and  the  fermentation  activity  of  the 
organism,  which  is  also  brisker  when  oxygen  is  excluded. 

LEUCONOSTOC   INDICUM. 

This  organism  is  the  cause  of  considerable  damage  to  the  Java  sugar 
industry.  According  to  Leisenberg  and  Zopf  the  only  difference 
between  this  organism  and  the  Leuconostoc  mesenteroides  is  a  slight 
difference  in  the  optimum  temperature  at  which  it  develops. 

ASCOCOCCUS    BILLROTHII. 

Found  by  Billroth  in  putrefying  meat  infusion. 

Microscopical  Appearances. — Small  cocci,  arranged  in  peculiar 
colonies,  which  form  a  creamy  layer  upon  the  surface  of  liquid  media, 
containing  numerous  small  spherical  or  oval  masses.  These  masses 
consist  of  a  jelly-like,  extremely  resistant  envelope,  from  10  to  15  (JL  thick  ; 
in  the  interior  of  the  envelope  one  or  more  masses  of  cocci  are  situated, 
from  20  to  70  /A  or  more  in  diameter.  The  cocci  are  closely  arranged 
and  united  by  a  firm  and  scanty  intercellular  substance. 

Biological  Characters.  —  Aerobic.  Grows  at  ordinary  room 
temperature.  Produces  a  strongly  alkaline  reaction  in  culture  media, 


THE  PHOSPHORESCENT  BACTERIA  225 

due  to  the  development  of  ammonia.  According  to  Cohn,  it  produces  a 
greenish-white  slimy  mass  upon  slices  of  beetroot,  and  in  the  juice  of 
sugar  beets  a  slimy  fermentation. 


THE   PHOSPHORESCENT   BACTERIA  (Photo-Bacteria 
Beyerinck). 

BACTERIUM   PHOSPHORESCENS   (FISCHER). 

Found  on  dead  meat  and  fish. 

Occurs  as  non-motile,  short  rods,  sometimes  in  zoogloea  ;  forms  no 
spores,  and  stains  by  the  Gram  method. 

Biological  Characters. — Facultative  anaerobe.  It  only  grows  on 
media  containing  chloride  of  sodium. 

In  Gelatine  Stab  Cultures  the  growth  is  usually  on  the  surface,  and  the 
gelatine  is  not  liquefied. 

Ferments  all  kinds  of  sugar. 

The  blue-green  phosphorescence  is  best  seen  on  cultures  on  dead 

fish,  meat,  and  sea-water. 

« 

BACTERIUM   PHOSPHORESCENS   PFLUGERI. 

Found    under    similar    conditions    to    the    above  Bacterium    phos- 

phorescens,   and   also    exhibits    identical    properties.  It    is,    however, 

longer,  more  slender,  and  does  not  so  frequently  form  zoogloea.     Causes 
fermentation  in  all  the  sugars  except  maltose. 

BACILLUS   ARGENTO   PHOSPHORESCENS  (KATZ). 

Found  by  Katz  in  Australia  in  sea-water  and  on  dead  sea  animals. 
There  are  three  varieties,  which  only  exhibit  a  slight  differentiation. 

They  occur  in  rods  0'6  to  0'8  by  2'5  /x,  are  motile,  and  stain  by  the 
Gram  method. 

The  culture  media  are  not  liquefied.  Yellowish  colonies  develop, 
exhibiting  a  silvery-white  phosphorescence  of  a  greenish  tint,  which  is 
however,  not  so  pronounced  as  in  the  aforementioned  varieties. 

BACILLUS   PHOSPHORESCENS   (GIARDI). 

Found  on  living  and  dead  crustaceous  animals.  It  is  pathogenic  for 
the  same,  especially  Talitrus  orchestia,  which  it  kills  in  six  to  nine  days, 
the  whole  body  of  the  animal  being  covered  with  greenish  phos- 
phorescent bacteria.  When  cultivated  on  ordinary  media  they  lose 

P 


226  SPECIAL  BACTERIOLOGY 

their  virulence,  but  regain  it  again  when  cultivated  on  fish  media. 
Morphologically  and  in  the  cultures  the  appearance  is  very  similar  to 
the  Bacterium  phosphorescens,  only  smaller  and  more  coccoid-like. 

BACILLUS   PHOSPHORESCENS   INDICUS   (B.   FISCHER). 

Cultivated  by  B.  Fischer  from  phosphorescent  sea-water  in  the  West 
Indies. 

Microscopical  Appearances. — Motile  rods,  twice  as  long  as  broad 
(0'6  to  0*8  by  2  fjC),  often  arranged  in  crooked  threads. 

Staining  Reactions. — Do  not  stain  by  the  Gram  method. 
Spore  Formation  absent. 

Biological  Characters. — Grows  under  aerobic  conditions  at  medium 
temperatures. 

On  Gelatine  Plates,  round,  bluish-green,  slowly  liquefying  colonies, 
which  later  become  granular  and  of  a  brownish  colour. 

In  Gelatine  Stab  Cultures,  funnel-shaped  liquefying  growth ;  in  the 
deeper  portion  of  the  medium  the  growth  is  limited. 

On  Agar  and  on  Potatoes  cooked  in  sea- water,  a  dirty  whitish 
coating  develops.  Blood  serum  is  liquefied.  The  blue  phosphorescence 
is  very  well  marked  in  cultures  on  dead  sea  animals,  sea-water,  and 
meat.  The  organism  is  non-pathogenic. 

BACILLUS   PHOSPHORESCENS   INDIGENUS   (FISCHER); 

Found  by  Fischer  in  Kiel  harbour.  It  is  very  similar  to  the  B. 
phosphorescens  indicus,  only  gelatine  medium  is  liquefied  more  slowly, 
while  blood  serum  is  not  liquefied.  It  grows  at  lower  temperatures,  and 
causes  no  phosphorescence  on  meat. 


THERMOPHILIC   BACTERIA. 

Miquel  found  a  bacillus  in  the  Seine  in  1891;,  which  possessed  the 
faculty  of  growing  at  a  temperature  of  69°  to  70°  C.  In  1887,  Koch  and 
Globig  found  bacteria  in  the  surface  of  the  earth,  which  grew  between 
50°  and  70°  C.  M'Fadyean  and  Rabinowitsch  have  also  contributed  to 
our  knowledge  of  this  group  of  bacteria. 

The  Thermophilic  bacteria  are  mostly  bacilli  that  are  facultative 
anaerobes  and  non-pathogenic,  Most  of  the  forms  produce  spores  which 
exhibit  great  resistance.  All  the  various  forms  grow  at  a  temperature 
between  56°  and  58°  C.  A  few  forms  grow  at  68°  C.,  and  at  70°  C.  Globig 
observed  growth  only  exceptionally. 


BACILLUS  CAPSULATUS  227 

THE  DRUMSTICK  BACTERIA. 

(Ger.  Trommelschlagerbacillen. ) 

This  is  a  group  of  saprophytic  organisms  which  form  spores  at  the  end 
of  the  bacillus,  which  thus  acquires  the  form  of  a  drumstick.  (See  Photo- 
micrograph, Fig.  82«.)  These  bacilli  are  widely  distributed  organisms. 

Microscopical  Appearances. — Very  small  rods,  strongly  motile. 

Gelatine  Media  are  liquefied. 

Grether  describes  a  variety  that  is  non-motile,,  and  does  not  liquefy 
gelatine. 

BACILLUS  CAPSULATUS  (PFEIFFER). 

Pfeiffer  isolated  the  organism  from  a  purulent  exudate  found  in 
the  peritoneal  cavity  of  a  dead  guinea-pig,  also  present  in  the  blood. 

Microscopical  Appearances. — It  is  a  plump  bacillus  with  rounded 
ends,  possessing  a  well-defined  ovoid  capsule. 

Motility. — Non-motile. 

Spore  Formation  has  not  been  confirmed. 

Staining  Reactions. — The  reaction  with  the  Gram  method  is 
negative  ;  the  capsules  are  easily  demonstrated  when  stained  by  Johne's 
method.  (See  Techniqne,  §  22.) 

Biological  Characters. — Facultative  anaerobe  growing  on  the 
ordinary  nutrient  media,  better  at  37°  C.  than  at  ordinary  room 
temperature. 

In  Gelatine  Stab  Cultures  a  slimy,  white,  nail-formed  growth  develops, 
and  an  inodorous  gas  is  formed ;  the  gelatine  is  not  liquefied. 

On  the  Surface  of  A  gar  it  forms  a  thick,  moist,  white  viscid  coating. 

On  Potatoes,  a  yellowish-white  viscid  covering. 

Pathogenesis. — White  and  house  mice  inoculated  subcutaneously 
die  in  from  two  to  three  days.  The  spleen  of  the  dead  animals  is  found 
greatly  enlarged,  and  the  bacilli  are  present  in  the  blood  and  organs, 
with  well-defined  capsules.  Guinea-pigs,  pigeons,  and  rabbits  are  also 
susceptible,  guinea-pigs  and  pigeons  only  by  intraperitoneal  infection, 
and  rabbits  only  when  large  quantities  of  the  culture  are  introduced  intra- 
venously. The  bodies  of  the  dead  animals  undergo  putrefactive  changes 
very  quickly.  The  blood  and  tissue  juices  exhibit  a  stringy  consistence. 


228  SPECIAL  BACTERIOLOGY 

BACILLUS  MEGATERIUM. 
(De  Barry.) 

Found  in  the  earth,  air,  and  on  the  leaves  of  cooked  cabbages. 

Microscopical  Appearances. — Very  long  (sometimes  10  p  and 
2*5  /x  thick),  slightly  bent  bacilli  with  round  ends.  Involution  forms 
often  present. 

Motility. — Slightly  motile ;  flagella  four  to  eight,  arranged  on  the 
sides  (peritricha). 

Spore  Formation. — Endogenous,  with  spores  nearly  as  long  as  the 
cells.  (For  Photomicrograph  of  rods  and  spores,  see  Fig.  83.) 

Biological  Characters. — Strongly  aerobic ;  optimum  temperature, 
20°  C. 

On  Gelatine  Plates,  kidney  or  sickle-shaped  granular  colonies,  that 
liquefy  the  medium  slowly. 

On  Agar,  a  whitish  coating. 

On  Potatoes,  a  thick  greyish-yellow  coating. 

On  all  the  media  it  forms  a  slimy  mass ;  and  according  to  Giinther, 
should  be  classified  with  the  capsule  bacteria. 

BACTERIUM   ZOPFII. 

Found  by  Kurth  in  the  intestinal  contents  of  a  chicken  in  1883. 
It  has  also  been  found  in  water  and  faeces.  Gunther  found  it  in 
sausages  in  1897. 

Microscopical  Appearances. — Short,  plump  bacilli,  2  to  5  //.  long 
and  0*75  to  1  ft  broad,  forming  long,  short-jointed  chains. 

Motility.— Motile. 

Staining  Reactions. — By  the  ordinary  methods  and  also  by  the 
Gram  method. 

Biological  Characters. — Aerobic ;  at  37°  C.  the  growth  is  not  so 
luxuriant  as  at  lower  temperatures. 

On  Gelatine  Plates. — Examined  with  a  low  power  the  colonies  consist 
of  long  threads  coiled  up  like  tangled  balls. 

In  Gelatine  Stab  Cultures,  the  growth,  which  takes  place  only  on  the 
surface,  consists  of  a  coating  of  finely-arranged  radiating  threads.  Old 
stab  cultures  are  inodorous. 

On  A  gar,  at  37°  C.,  a  thin  greyish  coating  develops. 

In  Bouillon,  at  37°  C.,  the  growth  is  hardly  visible. 

Milk  is  not  altered. 


FIG.  83.— B.  Megaterium  and  Spores  from  a  culture.    Fuchsin.     X  1000. 


FIG.  84.— Cladotlirix  found  in  water.    Cover-glass  specimen  from 
gelatine  culture.    Fuchsin.     X  1000. 


[T.  Bon-hill,  F.R.C.V.S.,  Photo.,  Edinburgh, 


THE  CLADOTHRICES  229 

The  reaction  of  Grape  and  Milk  Sugar  Media  is  not  changed,  and  no 
gas  is  formed. 

Indol  is  not  formed. 
Non-pathogenic. 

THE    CLADOTHRICES. 

These  organisms  are  found  in  water,  and  consist  of  colourless 
bacteria,  which  do  not  contain  sulphur  grains ;  arranged  in  threads. 
The  principal  characteristic  of  this  group  is  thejalse  branching  of  the 
threads  or  pseudo-ramification.  Three  forms  of  Gladothrix  are  well 
known. 

CLADOTHRIX   DICHOTOMA  (R  COHN). 

Found  in  both  standing  and  running  waters,  which  are  more  or  less 
rich  in  organic  substances.  It  is  frequently  associated  with  the 
Beggiatoa.  It  occurs  in  stationary  tufts,  1  to  3  mm.  high,  and  free  in 
floating  flakes.  From  the  point  of  the  Cladothrix  threads  individual 
elements  (Rodgonidia)  become  detached  and  float  round  free  for  a  time, 
then  become  stationary,  and  develop  into  fresh  threads.  The  individual 
threads  possess  a  distinct  sheath.  Artificial  cultivation  is  very  difficult. 
In  solutions  of  extract  of  meat  a  thin  film  forms,  which  extends  over  the 
surface  of  the  media  and  the  walls  of  the  tube.  In  meat  extract  gelatine, 
ramifying  colonies,  causing  very  slight  liquefaction  of  the  medium,  develop. 
(For  Photomicrograph  of  Cladothrix  cultivated  from  drinking  water,  see 
Fig.  84.) 

CLADOTHRIX    INTRICATA   (RUSSELL). 

Found  in  sea  slime  in  the  Gulf  of  Naples.  It  is  always  free,  and 
develops  no  sheath.  It  forms  no  tree-like  ramifications,  but  an  interlaced 
mass  of  threads.  In  certain  threads  the  pseudo-ramification  occurring 
in  C.  dichotoma  takes  place.  The  fresh  threads  are  homogeneous,  and 
when  stained  the  large  bacilli  of  which  they  are  formed  become  distinctly 
visible.  When  free  those  bacilli  become  actively  motile,  and  develop 
spores  which  are  not  thicker  than  the  threads.  It  is  easily  cultivated 
artificially. 

On  Gelatine  Plates,  mould-like,  quickly  liquefying  colonies  develop, 
which  under  a  low  power  appear  to  consist  of  a  tangled  mass  of  threads. 

In  Gelatine  Stab  Cultures  radiations  are  given  off  from  the  growth, 
which  become  shorter  towards  the  bottom  of  the  stab. 

On  Agar,  a  whitish  coating  develops,  from  which  projections  penetrate 
the  medium. 


230  SPECIAL  BACTERIOLOGY 

On  Potatoes,  a  whitish  coating. 

In  Bouillon,  a  gelatinous  sediment. 

CLADOTHRIX   OCHRACEA    (WINOGRADSKY). 

Found  in  water  containing  iron.  Very  like  the  C.  dichotoma.  For 
its  growth  the  water  must  contain  carbonate  protoxide  of  iron.  By 
oxidation  an  oxyhydrate  of  iron  is  formed,  which  is  deposited  in  the 
sheath  and  not  in  the  threads  of  the  organism.  In  water  and  under  the 
cover-glass,  cultures  can  be  obtained  by  the  addition  of  iron  salts.  In 
ordinary  media  the  results  are  negative. 


BEGGIATOA. 

Found  in  water  containing  sulphuretted  hydrogen. 

Microscopical  Appearances. — White  threads  without  any  distinct 
cell  membrane,  enclosing  in  their  interior  dark  grains  of  sulphur,  formed 
by  the  oxidation  of  the  sulphuretted  hydrogen.  If  a  microscopical 
specimen  is  prepared  and  the  sulphur  granules  dissolved  by  adding  alcohol 
or  bisulphide  of  carbon,  a  distinct  system  of  transverse  septa  will  be 
visible  in  the  threads. 

Biological  Characters. — It  can  be  cultivated  in  water  containing 
sulphuretted  hydrogen  (also  under  the  cover-glass),  where  active  motility 
and  slow  growth  is  observed.  Flagella  have,  however,  not  yet  been 
demonstrated.  Should  the  supply  of  sulphuretted  hydrogen  be 
interrupted,  then  the  small  grains  of  sulphur  in  the  threads  are 
gradually  changed  into  sulphuric  acid,  when  the  threads  appear 
completely  homogeneous.  In  time  degenerative  changes  occur,  vacuoles 
being  formed.  The  method  by  which  the  Beggiatoa  increase  is  not 
absolutely  known. 


BACTERIA  FOUND  IN  LEGUMINOUS  NODULES. 

The  earliest  description  of  the  leguminous  nodules  is  by  Malpighi  in 
his  book  published  in  1687,  in  which  he  referred  to  them  as  galls,  i.e. 
diseased  excrescences.  In  1853,  Treviraiius  considered  the  nodules 
normal  growths.  In  1866,  Woronin  discovered  that  there  were 
numerous  entirely  closed  cells  filled  with  living  bacteria  within  these 
nodules.  In  1879*  Frank  showed  that  nodule  formation  did  not  occur 
when  the  plants  were  grown  in  sterilized  soil,  thus  proving  that  the 
co-operation  of  soil  bacteria  was  a  necessary  factor.  In  1888,  the  true 


BACILLUS  RADICICOLA  231 

nature  of  these  nodule  bacteria  was  established  without  doubt  when 
Beyerinck  isolated  them  from  the  nodules  and  cultivated  them  further 
on  artificial  media.  These  bacteria  are  now  looked  upon  as  the 
generators  of  the  nodules,  by  means  of  which  the  Leguminosce  are  enabled 
to  absorb  nitrogen  from  the  air  and  elaborate  it  into  nitrogenous 
compounds,  albumen,  etc.  The  bacteria  are  situated  in  the  cells  of  the 
inner  layer  of  the  nodule,  which  is  known  as  the  bacteroidal  tissue,  and 
under  the  influence  of  the  surrounding  protoplasm  are  modified  into 
involution  forms  termed  Bacteroids,  rich  in  albumen  and  no  longer  capable 
of  reproduction. 

For  further  information  on  this  subject,  the  reader  is  referred  to  an 
interesting  article  on  'The  Bacteria  of  Soil,  with  special  reference  to 
Soil  Inoculation,'  by  R.  Stewart  MacDougall,  in  the  Transactions  of  the 
Edinburgh  Botanical  Society,  July  1897. 


BACILLUS   RADICICOLA   (BEYERINCK). 

Found  in  young  root  nodules,  also  in  the  tissues  of  plants. 

Microscopical  Appearances.—  In  cultures  it  occurs  as  large  rods 
1  to  4>  //,  long,  and  in  small  clusters  or  rovers  0'18  to  0'9  p-  The  large 
rods  frequently  exhibit  knotted,  irregular,  fork-shaped  or  three-armed 
bodies.  The  bacteria  in  the  nodules  exhibit  a  similar  morphology.  The 
rovers  belong  to  the  smallest  of  known  bacteria,  and  can  escape  through 
the  pores  of  a  Chamberland  filter. 

Motility. — Motile,  especially  the  rovers,  which  sometimes  escape  from 
the  parent  colonies,  and  found  a  daughter  colony  at  a  distance  in  the 
gelatine. 

Spore  Formation  absent. 

Staining  Reactions. — Sections  of  the  nodules  are  best  stained  in  a 
solution  of  equal  parts  of  fuchsin  and  methyl-violet  in  1  per  cent,  acetic 
acid.  The  plasmal  contents  and  membrane  of  the  nodule  cells  are 
coloured  blue,  the  bacteria  of  the  infection  threads  red,  whilst  the 
membrane  of  the  latter  remains  uncoloured. 

Biological  Characters.  —  These  organisms  grow  under  aerobic 
conditions  on  ordinary  gelatine,  but  very  slowly.  The  best  medium  is  a 
decoction  of  Papilionaceae  leaves  or  stalks,  with  7  per  cent,  gelatine,  J 
per  cent,  asparagin,  and  J  per  cent,  cane-sugar  added.  The  developing 
colonies  are  semi-circular,  whitish  or  slightly  clouded.  The  larger 
colonies  are  watery,  while  the  smaller  ones  are  solid  and  adhere  in  one 
piece.  According  to  Beyerinck,  the  various  species  of  Leguminosae 
exhibit  a  difference  in  the  form  of  the  colonies  and  the  bacteria.  Kruse 
considers  that  they  are  probably  only  varieties  of  one  species. 


232  SPECIAL  BACTERIOLOGY 

RHIZOBIUM   LEGUMINOSARUM   (FRANCE.) 

Franck  obtained  cultures  of  this  organism  in  liquefied  drops  of 
gelatine  under  microscopic  control,  and  also  on  plates.  According  to 
the  same  author,  the  root  bacteria  occur  only  in  the  clusters  (0*9  to 
1'3  n  in  length),  oval  or  rod-shaped,  which  later  form  zooglrea.  The 
gelatine  is  liquefied  slowly.  The  morphology  is  the  same  in  all  the 
Leguminosae. 

According  to  Franck  one  could  at  the  most  accept  different  varieties 
or  nutrition  modifications. 

BACILLUS  TUBERIGENUS   (VON  GONNERMANN.) 

Found  in  the  root  nodules  of  the  lupine  bean,  etc.  Von  Gonner- 
mann,  with  a  specially  prepared  lupine  peptone  gelatine,  isolated  ten 
different  species  of  bacteria,  two  cocci,  the  Bacillus  fluorescens  non- 
liquefaciens,  and  seven  bacilli  which  the  author  named  Bacillus  tuberi- 
genus,  Nos.  1  to  7.  When  sterilized  earth  was  inoculated  with  Nos.  3 
and  5,  the  formation  of  typical  root  nodules  took  place. 

BACILLUS   TUBERIGENUS   (No.  3). 

Is  motile,  and  0*3  to  0'6  //.  in  size. 

On  Gelatine. — Well  defined,  yellowish-brown,  finely  granular  colonies 
develop  ;  the  gelatine  is  liquefied  quickly. 

On  Potatoes,  a  bright  reddish-brown  coating. 

BACILLUS   TUBERIGENUS   (No.   5). 

Is  non-motile  and  more  slender  than  No.  3  (0*25  to  2  /x). 

On  Gelatine  it  forms  colonies  resembling  those  of  the  Bacillus 
anthracis,  but  does  not  give  off  any  offshoots  into  the  surrounding  media. 

On  Potatoes  it  forms  yellow  prominent  drops. 

These  bacilli,  as  well  as  the  Bacillus  radicicola,  penetrate  the  root 
tissue  of  the  Leguminosae,  and  form  in  the  cells  irregular  masses  with 
offshoots  and  vacuoles,  and  as  such  possess  the  faculty  of  assimilating 
the  nitrogen  of  the  air.  Entire  clearness  over  the  importance  of  these 
bacteria  does  not  yet  exist,  nevertheless  they  appear  to  play  an  import- 
ant role  in  the  nourishment  of  plants,  especially  the  Leguminosae. 

THE   NITRIFYING   BACTERIA. 

According  to  Winogradsky  the  many  species  of  nitrifying  bacteria 
can  be  classified  into  two  sharply  divided  sub-groups, — Nitroso-bacteria 
and  Nitro-bacteria.  The  Nitroso-bacteria  oxidize  ammonia  to  nitrous  acid. 


THE  N1TRO-BACTERIA  233 

During  the  oxidation  processes  the  bacteria  are  protected  from  injury 
by  the  presence  of  bases  which  take  up  the  acids  with  which  the 
ammonia  was  initially  combined,  and  also  neutralize  the  resulting 
nitrous  or  nitric  acid.  Calcium  carbonate  performs  this  function  excel- 
lently in  the  soil.  Free  alkali  is  unsuitable  here  for  the  fixation  of  the 
acids,  because  if  present  in  quantity  it  would  be  injurious  to  the  bacteria. 
In  artificial  cultures  Winogradsky  replaces  the  calcium  carbonate  by 
magnesium  carbonate  (Lafar's  Tech.  Mycology,  §  204-). 


THE    NITRO-BAOTBRIA. 
NITROSOMONAS   EUROP^EA   (WINOGRADSKY). 

Found  in  all  samples  of  European,  African,  and  Japanese  earth 
examined. 

Microscopical  Appearances. — They  occur  in  short  chains  of  three 
to  four  individuals. 

Spore  Formation  absent. 
Motility  present  (Monotricha). 

Biological  Characters. — For  method  of  isolating  this  organism 
see  Technique,  §  156. 

On  Silicic  Acid  Media  the  colonies  are  at  first  compact,  with  a  sharp 
contour,  brownish  colour,  sometimes  resembling  a  spindle  with  blunt 
ends.  In  ten  to  fourteen  days,  round,  clear,  unstained  masses,  with 
irregular  offshoots,  consisting  of  motile  monas,  extend  from  the  growth. 

In  Fluid  Cultures,  when  quiescent,  they  collect  in  zooglcea,  forming  a 
sediment  especially  around  the  precipitated  carbonate.  In  seven  days  or 
less  the  fluid  becomes  cloudy,  and  in  twenty-four  to  forty-eight  hours  the 
motile  monas  again  sink  to  the  bottom.  The  process  of  growth  and  the 
nitrite  formation  is  now  finished. 

NITROSOMONAS    JAVANIENSIS   (WINOGRADSKY). 

Found  in  Java  earth,  and  very  similar  to  the  above  organism. 

NITROSOCOCCUS     BRAZILIENSIS. 

Found  in  the  soil  of  Campinas,  Brazil.  They  do  not  form  zooglcea, 
and  possess  no  cilia,  and  attain  a  diameter  of  2  /x.  The  species  grown 
from  Melbourne  soil  is  indistinguishable  from  that  found  in  Brazil ; 
while  that  obtained  from  Quito  (Ecuador)  is  a  coccus,  1*5  to  1*7  />t  in 
diameter. 


234,  SPECIAL  BACTERIOLOGY 


NITRO-BACTERIA   (WINOGRADSKY). 

This  organism  was  isolated  from  Quito  earth,  and  forms  nitrates 
out  of  nitrites. 

Microscopical  Appearances. — Very  small  rods,  0-2  to  0'25  by  0-5  /*. 
Motility. — Non-motile. 

Biological  Characters. — On  Silicic  Acid  Plates  they  develop  in 
lenticular  or  club-shaped  colonies. 

In  Fluid  Media  it  develops  in  the  form  of  thin  films,  firmly  adherent 
to  the  walls  and  bottoms  of  the  flasks  —  there  is  no  cloudiness. 
Burri  and  Stutzer  have  with  the  assistance  of  silicic  acid  plates  isolated 
a  nitrate  builder  in  European  earth,  which  is  distinguished  from  the 
above  Winogradsky  organism  by  being  somewhat  larger  on  solid  media, 
motile,  and  transferable  to  ordinary  nutrient  media  (gelatine  and 
bouillon).  When  grown  on  the  latter  medium  and  again  returned  to  a 
nitrate  solution,  have  mostly  exhibited  the  remarkable  faculty  of  pro- 
ducing nitrification. 

Winogradsky  ascertained  by  comparative  investigations  that  the 
nitroso-bacteria  are  the  more  active  of  the  two. 

Both  nitroso-  and  nitro-bacteria  are  always  present  in  the  soil,  the 
latter  immediately  oxidizing  the  nitrous  acid  generated  (from  the 
ammonia  salts)  by  the  former. 

Whether  nitrification  commences  in  the  dung-heap  or  in  the  field  is 
dependent  on  various  circumstances.  It  takes  place  whenever  a 
sufficient  amount  of  ammonia  salts  has  been  produced  by  the  fermenta- 
tion of  urea,  provided  there  is  a  free  access  of  air.  H.  ImmendorfF 
showed  that  in  the  outer  layers  of  manure  heaps  (especially  horse  dung) 
nitrous  acid  is  produced  briskly  in  a  few  days.  On  account  of  the 
formation  of  easily  lixiviable  nitrates,  which  may  moreover  expose  the 
material  to  wasteful  reduction  processes,  endeavours  should  be  made 
to  minimise  the  aeration  of  the  manure  by  battening  the  heaps  well 
down. 

It  is  well  known  that  the  soil  has  no  power  of  fixing  nitrates,  as 
according  to  P.  Deherain  and  others  a  certain  portion  of  the  added 
saltpetre  invariably  escapes  in  the  drainage  water,  so  that  more  has  to 
be  added  to  the  soil  than  is  recovered  in  the  crop. 

In  manuring  with  salts  of  ammonia  no  such  waste  occurs,  as  they  are 
fixed  by  the  soil  and  protected  from  wasteful  lixiviation,  the  nitrifying 
bacteria  then  oxidizing  the  ammonia  and  supplying  the  plant  with 
nitrites  according  to  its  requirements  (Lafar's  Tech.  Mycology,  §§  206, 
208). 


PART    IV. 

THE  HYPHOMYCETES,  OR  MOULD  FUNGI. 

ACHORION   SCHONLEINIL 

This  fungus,  the  cause  of  favus  in  man,  horses,  cattle,  dogs,  cats, 
rabbits,  and  mice,  was  discovered  by  Schonlein  in  1839.  It  is  found 
in  the  so-called  favus  crusts.  According  to  Unna,  there  are  nine 
varieties  of  favus.  The  disease  is  most  frequently  observed  on  a  head 
possessing  hair,  although  it  is  also  found  on  skin  devoid  of  hair.  It 
also  attacks  the  nails  (Onychomycosis),  the  parasite  being  located 
between  the  cells  of  the  epidermis  and  the  corium.  Kaposi  also 
mentions  a  case  of  favus  universalis. 

Microscopical  Appearances. — The  mycelium  consists  of  branched 
radiating  hyphae.  Some  of  the  hyphae  swell  at  their  free  ends, 
becoming  club-shaped,  while  others  give  off  lateral  buds  containing 
Krals'  so-called  yellow  bodies,  which  rupture,  allowing  their  contents  to 
escape  as  free  bodies.  When  this  takes  place,  moss-like  offshoots 
develop  in  the  form  of  dense  twisted  threads.  Later,  the  individual 
threads  break  up  into  cell-like  oval  structures. 

Biological  Characters. — To  obtain  the  fungus  in  pure  cultures, 
the  favus  crusts  are  mixed  with  sterile  sillic  acid,  reduced  in  a  sterile 
mortar,  and  plate  cultures  instituted  with  the  mixture.  The  growth 
takes  place  at  both  room  and  incubator  temperature  on  all  the  nutrient 
media,  usually  under  the  surface,  because  only  a  few  air-hyphae  are 
formed.  In  the  beginning,  from  the  periphery  of  the  white  growth, 
which  later  becomes  yellow,  fine  radiating  offshoots  penetrate  the  body 
of  the  substratum  of  the  medium.  (See  Photomicrograph,  Fig.  86.) 

On  Gelatine  Plates,  white,  stellate,  quickly  liquefying  colonies,  with 
thick  centres. 

In  Gelatine  Stab  Cultures  a  coating  forms  on  the  surface,  the  under- 
growth being  of  a  yellow  colour. 

On  Agar  a  puckered  whitish  coating,  the  under  surface  being  yellow. 
(See  Photograph,  Fig.  85.) 


236  THE  HYPHOMYCETES 

Blood  Serum  is  the  only  medium  on  which  spores  are  developed,  the 
most  favourable  temperature  being  30°  C. 

Pathogenesis.  —  Artificial  infection  can  only  be  produced  with 
material  containing  spores. 

TINEA  GALLI   (SCHUTZ). 

The  chicken  favus  is  due  to  this  fungus,  which  attacks  the  comb, 
wattles,  and  side  of  the  throat.  Round  spots  appear,  which  usually 
become  confluent,  spreading  to  the  neck,  breast,  and  body. 

Microscopical  Appearances. — The  fungus  consists  of  a  mycelium 
formed  of  pointed  and  often  branched  threads  of  variable  dimensions, 
which  often  have  small  wart-like  pedunculate  projections,  while  other 
joints  are  club-shaped  and  sometimes  found  free,  and  here  and  there 
'fringed  with  offshoots.  In  some  cases,  fine  offshoots  can  be  seen  on 
the  sides  of  the  mycelium  bearing  one  or  two  club-shaped  grey-coloured 
bodies. 

Biological  Characters. — On  Gelatine  a  whitish  growth  develops  ; 
the  gelatine  is  liquefied,  acquiring  a  reddish  colour. 

It  also  grows  on  potatoes  and  bread  paste,  the  best  temperature 
being  about  30°  C. 

Pathogenesis. — The  characteristic  symptoms  are  produced  in 
chickens  with  pure  cultures  ;  while  mice,  rabbits,  and  various  other 
experiment  animals  remain  unaffected.  According  to  the  conclusions 
of  MM.  Constantine  and  Subrayes,  three  distinct  parasites  are  the  cause 
of  favus  in  man,  the  dog,  and  the  fowl.  Human  favus  is  nearly  related 
to  that  of  the  dog,  but  distinguished  from  the  latter  by  its  appearance 
in  cultures  and  by  the  invariable  structure  of  its  mycelium  and  by  its 
colour. 

TRICHOPHYTON   TONSURANS. 

This  fungus  is  found  in  the  epithelial  scales  in  herpes  tonsurans. 

Microscopical  Appearances. — The  single  mycelial  threads  are 
distinctly  septate,  and  from  some  of  them  conidia  are  given  off  in  a 
similar  manner  to  the  Oidium  lactis.  (For  Photomicrograph  of  this 
fungus,  see  Fig.  88.) 

Biological  Characters. — It  grows  at  room  temperature,  while 
the  optimum  temperature  is  30°  C. 

On  Gelatine  Plates. — Semi-globular ;  white,  later  yellow,  liquefying 
colonies. 

In  Gelatine  Stab  Cultures,  white  coating,  which  floats  on  the  surface 
when  liquefaction  occurs. 


FIG.  85.— Achorion   Schiiu- 
leinii.     Agar  culture. 


FIG.  87.— Tricophytou  Toi 
surans.     Agar  culture. 


SSlSmW-fe: 


FIG.  86.— Achorion  Schonleiuii.    Section  of  an  agar  culture. 
Fuchsin.     X  600. 


[T.  Bou-klU,  F.n.C.l'.X.,  Photo.,  Edinburgh,  1898, 


THRUSH  237 

On  Agar  it  forms  white  tufts  somewhat  puckered  (see  Photograph, 
Fig.  87),  while  the  deeper  underlying  portions  are  of  a  yellowish  colour. 

On  Blood  Serum,  white  tufts ;  the  medium  is  liquefied. 

On  Potatoes,  a  slow  growth. 

In  contradistinction  to  other  fungi,  the  cultures  retain  their  vitality 
for  a  long  time. 

Pathogenesis. — The  artificial  production  of  Herpes  tonsurans  with 
material  containing  gonidia  has  taken  place. 

Sabouraud  distinguishes  two  groups  of  trichophyton  which  he  classi- 
fies as  Botrytis  tonsurans,  characterised  by  the  grape  form  arrangement 
of  the  fruit.  Both  groups  are  distinguished  by  the  size  of  their  spores, 
and  according  to  Sabouraud,  are  named  Trichophyton  microsporon  and 
megalosporon. 

The  first  form  affects  only  hairy  places,  and  its  spores  are  3  //,  in 
diameter.  It  causes  the  severe  affection  in  children  known  as  the 
Maladie  de  Gruby. 

The  second  form  has  spores  from  7  to  8  /x  in  diameter,  and  causes 
in  adults  a  trichophyton  affection  of  the  beard  as  well  as  of  bald  parts 
of  the  body.  According  to  Sabouraud  the  trichophyton  affecting  man 
and  animals  exhibit  well-marked  morphological  and  clinical  differentia- 
tion ;  and  furthermore,  he  states  that  the  trichophyton  microsporon  when 
grown  on  potatoes,  in  contradistinction  to  all  other  varieties  which  die 
after  three  weeks  on  that  media,  continues  to  grow  slowly,  and  after 
three  months  further  cultures  can  be  instituted  in  fresh  media  from  the 
original. 

THRUSH. 

This  disease  is  caused  by  a  fungus,  the  Old-turn  albicans,  and  occurs 
on  all  mucous  membranes  with  squamous  epithelium,  especially  in  the 
mouths  of  infants,  where  it  is  manifested  by  white  patches. 

Microscopical  Appearances. — Sometimes  mycelial  threads  are 
present,  and  at  other  times  round  or  oval  conidia  like  yeast  cells. 

Biological  Characters.  —  Strongly  aerobic  ;  optimum  tempera- 
ture, 37°  C. 

On  Gelatine  Plates,  white,  non-liquefying  colonies. 

On  Gelatine  Stab  Cultures,  yellow-white  grains,  with  processes  extend- 
ing into  the  medium. 

On  Agar,  a  yellowish-white  growth. 

On  Potatoes,  a  thick  white  coating,  which  frequently  is  observed  to 
consist  of  small  clusters. 

On  Bread  Paste,  a  thin  white  coating. 

In  Media  containing  Sugar  or  Acid  Media  it  grows  more  in  the  form 
of  buds  or  yeast-like  cells. 


238  THE  HYPHOMYCETES 

In  Alkaline  or  Media  deficient  in  Sugar  it  grows  more  in  the  form  of 
mycelial  threads. 

Pathogenesis. — Besides  being  found  in  infants'  mouths,  it  has  also 
been  observed  in  adults  in  the  oesophagus,  middle-ear,  trachea,  and  in 
both  nasal  cavities  in  a  young  man  after  a  severe  attack  of  influenza. 
Pathogenic  for  rabbits  when  inoculated  intravenously,  the  fungus  be- 
coming localized  in  the  internal  organs. 

OIDIUM   LACTIS. 

Found  in  sour  milk,  on  bread,  and  decayed  fruit.  On  cream  the 
colonies  can  be  recognised  by  transmitted  light  as  faintly  yellow 
round  spots. 

Microscopical  Appearances. —  Single  mycelial  threads  give  oft 
cylindrical  joints  like  yeast-cells  (oidien),  which  towards  their  extremities 
become  gradually  shorter,  resembling  conidia.  The  mycelium  consists 
of  septate  branched  threads  of  various  thickness.  (For  Photomicrograph 
of  this  fungus,  see  Fig.  89). 

Staining  Reactions. — Stains  easily  with  the  ordinary  aniline  stains  ; 
but  dried  specimens,  owing  to  the  heating  in  their  preparation,  cause 
shrinking  and  alteration  in  the  form  of  the  oidien. 

Biological  Characters. — It  grows  well  on  all  kinds  of  nutrient 
media,  especially  when  the  reaction  is  slightly  acid,  at  both  room  and 
incubator  temperature.  The  optimum  temperature  is  15°  to  20°  C. 

On  Gelatine  Plates  a  white,  long,  hairy,  mycelial  growth  develops, 
which  covers  the  plate  but  does  not  liquefy  the  medium. 

On  Agar  the  growth  at  first  is  delicate,  but  later  a  viscous  yellowish- 
white  coating  forms. 

On  Potatoes,  a  whitish  growth. 

On  Milk  a  skin  is  formed  on  the  surface. 

Sugar  is  fermented  and  Albuminoids  decomposed. 

Non-pathogenic. 

PENICILLIUM   GLAUCUM. 

This  fungus  is  of  universal  occurrence.  It  is  found  on  jam,  cheese, 
decaying  fruit,  and  on  barley  when  it  is  lying  on  the  malt-house 
floor.  It  also  occurs  on  the  walls  and  ceilings  of  rooms.  In  the 
young  stage  it  consists  of  whitish  tufts,  which  later  acquires  a  green 
colour,  due  to  the  formation  of  myriads  of  spores.  The  free  spores 
are  covered  externally  with  a  fatty  layer  which  protects  them  from 
moisture. 

Microscopical  Appearances. — A  mycelium  is  formed  from  which 


Fio.  88.-Tricophyton  Tonsurans,  from  agar  culture.     Unstained.     X  350. 


Fio.  89.— Oidium  Lactis,  from  gelatine  culture.     X  1000. 


I  V.  I'.nHdll,  F.R.C.V.H.,  Photo.,  Edinburgh,  1898. 


THE  ASPERGILLI  239 

numerous  hyphae  are  given  off.  Erect  filaments,  or  aerial  kyphce,  also 
extend  upwards,  shaped  like  a  broom,  and  bearing  at  the  end  of  their 
branches  the  spores.  (See  Photomicrograph,  Fig.  90.) 

Vitality  of  the  Spores.  —  The  spores  will  germinate  at  any 
temperature  between  2°  and  43°  C.  22°  to  26°  C.  is  the  most  favourable, 
while  according  to  Pasteur  dry  spores  withstand  a  temperature  of  108° 
C.,  but  are  soon  killed  when  immersed  in  boiling  water. 

Biological  Characters. — It  grows  best  at  room  temperature  on 
any  of  the  ordinary  media.  It  is  also  able  to  propagate  itself  sexually 
when  placed  under  certain  conditions,  especially  the  absence  of  oxygen. 

It  is  non-pathogenic. 

The  green  mould  growing  in  the  cracks  of  Roquefort  cheese  is  due  to 
this  fungus,  which  consumes  the  acid  produced  by  the  lactic  acid 
bacteria,  thus  retarding  the  development  of  albumin  -  degrading 
organisms. 

Experience  having  shown  the  favourable  action  of  this  fungus,  it  is 
grown  on  bread,  which  is  dried,  powdered,  and  mixed  between  the 
separate  layers  of  the  sliced  curd.  In  Edam  cheese  an  organism  known 
as  the  Streptococcus  Hollandicus  is  mixed  with  the  milk  before  it  is 
made  into  cheese. 


THE   ASPERGILLI. 

From  the  mycelium  individual  threads  pass  upwards  (air-hyphce),  and 
swelling,  form  clubs  without  dividing.  This  swelling  of  the  conidia  or 
fruit-bearers  is  surrounded  by  a  mass  of  cylindrical  cells,  which  at  their 
distal  ends  or  extremities  produce  chains  of  spherical  spores  or  conidia. 
Out  of  each  spore  a  new  fungus  can  develop.  When  highly  nourished, 
another  method  of  fructification  takes  place.  Some  of  the  terminal 
branches  of  the  mycelium  become  twisted  like  a  spiral,  and  are  known  as 
the  (  Carpogonium '  ;  from  the  same  thread  branches  grow  towards  the 
carpogonium,  one  of  which  becomes  fused  with  the  terminal  portion  of 
the  carpogonium,  known  as  the  '  Ascogonium '  ;  while  others,  the 
'  Pollinodia/  ramify  around  the  carpogonium  like  a  capsule, — the  whole 
organ  being  called  a  perithecium.  The  ascogonium  divides  rapidly  into 
a  number  of  oval  tubes,  inside  of  which,  by  endogenous  division,  small, 
round,  eight-spored  asci  develop.  The  aspergilli  grows  best  on  bread 
paste  and  acid  media,  beer- wort  gelatine,  agar,  and  potatoes. 

Asp.  Nidulans. — Found  on  bread  ;  light  green  tufts.  Air-hyphae 
present,  especially  in  old  cultures  which  are  sometimes  coloured  light 
red.  Branched  sterigmen ;  optimumt  emperature,  40°  C.  On  potatoes 
and  bread  it  forms  a  reddish-brown  pigment,  which  penetrates  the 
medium.  The  pathogenic  properties  are  the  same  as  the  A.  fumigatus. 


240  THE  HYPHOMYCETES 

Asp.  Niger. — Blackish-brown  tufts.  Branched  sterigmen  ;  optimum 
temperature,  34°  C.  (See  Photomicrograph,  Fig.  91). 

Asp.  Ochraceus. — Yellowish-red  to  dark-yellow  tufts.  Branched 
sterigmen. 

Asp.  Oryzse. — Found  on  rice  ;  at  first  flesh-coloured,  later  of  an 
ochre-yellow  colour  ;  changes  starch  and  dextrin  into  sugar.  Used  in 
the  preparation  of  the  Japanese  rice-spirit. 

Asp,  Repens. — Found  on  fruits  preserved  with  sugar.  Appears  at 
first  white,  later  as  greenish  tufts,  with  smooth  colourless  or  greenish 
spores. 

Asp.  Subfuscus. — Found  on  bread.     Appears  as  tufts,  yellowish  to 
black  in  colour.     Optimum  temperature,  37°.     Pathogenic. 
Asp.  Albus. — Whitish  tufts  and  branched  sterigmen. 

Asp.  Clavatus. — Greenish  tufts  and  club-shaped  fruit  bearers  and 
small  conidia. 

Asp.  Plavescens,  or  Plavus.  —  Found  in  bread,  greenish-brown 
tufts,  and  yellowish-brown  spores,  with  rugged  surface,  small  black 
sclerotien.  The  optimum  temperature  for  its  development  is  28°  C. 
The  pathogenic  properties  are  the  same  as  the  Asp.  fumigatus. 

Asp.  Fumigatus. — Found  in  the  trachea  and  bronchi  of  birds,  and 
also  on  bread ;  bluish-green  tufts,  which  later  acquire  a  bluer  colour ; 
very  small  smooth  spores ;  optimum  temperature,  37°  to  40°  C.  (For  Photo- 
micrograph of  this  fungus,  see  Fig.  92.) 

Pathogenesis. — When  rabbits  and  dogs  are  injected  intravenously  with 
the  spores,  death  occurs  in  about  twenty-four  hours.  In  all  the  organs, 
especially  in  the  substance  of  the  heart  and  kidneys,  the  fungi  can  be 
detected  in  small  clusters.  In  man  an  aspergillimycosis  also  occurs  in 
the  lungs,  auditory  canal,  and  on  the  cornea. 

Asp.  Glaucus. — Found  in  fruit,  cabbage,  and  damp  wooden  walls. 
Greenish-coloured  tufts  and  round  spores  with  rugged  surfaces ;  optimum 
temperature,  10°  to  15°  C.;  killed  at  25°  C.;  non-pathogenic. 

MICROSPORON  FURFUR. 

Found  in  the  scales  cast  off  in  pityriasis  versicolor.  When  these 
scales  are  treated  with  a  5  per  cent,  solution  of  caustic  potash,  and 
examined  microscopically,  short,  slightly  branched  mycelial  threads  are 
seen,  with  very  large  conidia  lying  together  in  clusters. 

Cultivations  have  not  yet  been  obtained. 

MICROSPORON   MINUTISSIMUM. 

Found  in  the  scales  cast  off  in  erythrasma,  and  similar  to  the  above, 
except  that  the  mycelia  and  conidia  are  here  excessively  fine. 


FIG.  90.-Penicillium  Glaucum,  from  gelatine  culture.    Unstained.     X  300. 


wfcw 


Fin.  91.— Aspergillus  Niger,  from  agar  culture. 
Unstained.     X  350. 


[T.  Boit'hill,  F.U.C.V.S.,  Photo.,  Edinburgh,  1898. 


FIG.  92. — Aspergillus  Fumigatus,  from  agar  culture. 
Unstained,     x  350. 


Fit;.  93. — Mucor  Corymbifer,  from  potato  culture. 
Unstained.     X  350. 


[T.  Bmohill,  F.R.C.r.S.,  Photo.,  Edinburgh,  1898. 


THE  MUCORS  241 


THE   MUCORS. 

Specially  characterised  by  aseptate  hyphae  springing  from  the 
mycelium.  In  these  hyphae  a  large  sporangium  develops  apically  and 
by  fission.  Inside  it  spores  are  produced,  which  later  are  liberated  by 
the  bursting  of  the  sporangium.  Under  some  conditions  proliferation 
occurs  by  conjugation  of  two  bodies  forming  what  is  called  the  zygo- 
spores. 

Mucor  Corymbifer. — Occurs  as  greyish  tufts.  The  hyphae  come 
off  from  the  mycelium  at  an  angle,  and  are  branched  with  colourless 
sporangia.  The  spores  are  small,  3  /*  to  2  /A.  Optimum  temperature, 
37°  C. 

Pathogenesis. — When  injected  into  the  veins  of  rabbits,  death  follows 
in  from  two  to  three  days.  The  clumps  of  fungus  are  mostly  found  in 
the  kidneys  and  lymph-follicles  of  the  intestinal  mucosa.  Dogs  are 
immune.  (For  Photomicrograph  of  this  fungus,  see  Fig.  93.) 

Mucor  Mucedo. — Found  on  horse  manure  as  a  whitish  growth,  like 
thistledown.  The  hyphae  are  1  to  13  cms.  in  length,  and  may  be  single 
or  branched  ;  crystals  of  oxalate  of  lime  occur  on  the  outside  of  the  spor- 
angia, which  later  are  coloured  brown  or  black.  (See  Photomicrograph, 
Fig.  94.)  Optimum  temperature,  37°  C. ;  non-pathogenic. 

Mucor  Pusillus. — Found  in  moist  bread,  and  occurs  in  tufts,  which 
are  at  first  white  and  get  grey.  The  mycelium  is  unusually  fine.  The 
sporangia  are  covered  with  a  spinous  membrane ;  optimum  temperature, 
45°  C. 

Pathogenesis. — Same  as  Mucor  corymbifer. 

Mucor  Ramosus. — Occurs  on  moist  bread  as  tufts,  at  first  white, 
and  later  brownish.  The  mycelium  is  very  much  branched,  and  has  long 
branched  air  hyphae  and  large  spores.  Optimum  temperature,  40°  C. 

Pathogenesis. — Same  as  Mucor  corymbifer. 

Mucor  Racemosus.  —  Found  on  sugar  and  starchy  substances. 
Has  many  delicate  fruit  hyphae  not  exceeding  1  to  5  cms.  in  length. 
Sporangia  are  yellow  or  yellowish-brown,  with  round  spores. 

Mucor  Stolonifer. — The  mycelium  has  branches  which  at  first 
ascend  and  then  descend,  and  are  covered  with  fine  root  hairs. 

The  sporangia  are  deep  black  and  tuberculated  with  brown  globose 
spores,  10  to  20  /A  in  diameter.  The  zygospores  are  blackish-brown. 

Mucor  Rhizopodiformis. — The  mycelium  is  at  first  quite  white,  and 
later  turns  greyish.  The  hyphae  are  twisted,  at  first  ascend,  and  then 
grow  down  again  into  the  medium,  where  they  get  covered  with  root 
hairs.  Spores  colourless  ;  diameter  5  to  6  fi, 

Q 


242  THE  HYPHOMYCETES 

Palhogenesis. — Same  as  Mucor  corymbifer,  but  is  distinguished  from 
it  by  the  pleasant  fruity  smell  of  the  cultures. 

The  mucors  are  the  lowest  members  of  the  vegetable  kingdom 
exhibiting  a  method  of  reproduction  which  is  universal  in  the  higher 
forms  of  plant  life.  Two  cells  are  concerned  in  this  process,  known  as 
the  male  and  female  elements,  and  the  process,  which  is  called  sexual 
reproduction,  only  takes  place  by  this  method  when  growing  on  a  solid 
substratum.  When  there  is  a  plentiful  supply  of  nourishment  at  hand 
reproduction  takes  place  asexually,  that  is,  by  spore  formation. 

FUSISPORIUM   MOSCHATUM   (KITASATO). 

This  fungus  is  described  by  Kitasato.  It  was  found  as  an 
accidental  growth  in  a  vegetable  infusion.  The  principal  charac- 
teristics are  the  formation  of  sickle-shaped  spores  (see  Photomicro- 
graph, Fig.  95),  and  an  odour  of  musk  given  off  by  the  cultures. 

Biological  Characters. — It  grows  only  at  room  temperature  on  all 
the  ordinary  media,  on  rice  paste,  and  infusion  of  peas. 

Gelatine  Media  are  liquefied  slowly ;  the  cultures  at  first  are  greyish- 
white,  eventually  becoming  rose  or  brick-red  coloured. 

In  preparing  and  mounting  microscopic  specimens  of  the  various 
fungi,  illustrated  by  the  accompanying  photomicrographs,  the  author 
adopted  the  following  method  : — 

1.  Hammer  out  a  piece  of  platinum  wire  quite  flat  at  the  point,  in 
the  shape  of  a  shovel. 

2.  Select  an  isolated  growth,  heat  the  shovel,  and  cut  through  the 
medium  a  short  distance  from  the  growth. 

3.  Push  the  platinum  spade  under  the  growth  and  transfer  the  mass 
en  bloc  to  a  clean  cover-glass. 

4.  Place  the  cover-glass  on  a  clean  slide,  heat  gently  over  the  flame 
(just  enough  to  melt  the  medium),  and  press  the  cover-glass  gently  to 
remove  superfluous  medium. 

By  this  process  the  fungus  is  mounted  on  the  medium  it  is  growing 
in,  and  the  character  of  the  growth  is  readily  observed.  In  making 
permanent  mounts  by  this  method,  a  drop  of  a  solution  of  formalin  is 
added  before  the  slide  is  heated. 


FIG.  94.— Mucor  Mucedo,  from  gelatine  culture,  shomng  Zygotes. 
Unstained,     x  350. 


FIG.  95. — Fusisporium  Moschatum,  from  potato  culture, 
showing  sickle-shaped  Spores.    Unstained,     x  350. 


[T.  Bowhill,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


PART    V. 

THE    BLASTOMYCETES,    OR   YEAST    FUNGI 

The  yeast  fungi  are  divided  into  two  groups : — 

(«)  Saccharomycetes  or  true  yeasts,  in  which  true  spore  formation 
occurs. 

(6)  Torulse,  in  which  no  spore  formation  has  been  observed. 

The  Blastomycetes  reproduce  themselves  by  gemmation  or  budding, 
which  distinguishes  them  from  the  schizomycetes  or  bacteria,  which 
reproduce  themselves  by  fission  or  simple  division.  From  the  hypho- 
mycetes  or  mould  fungi  the  blastomycetes  are  distinguished  by  being 
unicellular  and  by  asexual  reproduction. 

The  yeasts  employed  for  commercial  purposes  consist  of  a  mixture  of 
different  species.  We  are  indebted  to  the  researches  and  experiments 
of  Hansen  for  our  present  knowledge  of  the  various  changes  produced 
by  the  different  species  during  fermentation,  as  well  as  for  the  method 
of  isolating  them  by  means  of  plate  cultures  instituted  with  acid  or 
beer-wort  gelatine.  Observation  of  the  following  conditions  enables  the 
various  species  to  be  differentiated  during  development. 

1 .  The  temperature  at  which  ascospores  develop. 

2.  The  characters  of  the  film  or  zoogloea — mass  of  cells  forming  on 
the  surface  of  the  fluid  during  fermentation. 

3.  The  changes  produced  in  the  various  kinds  of  sugar. 

4.  Whether  the  variety  under  observation  causes  a  top  or  bottom 
fermentation. 

A  surface  or  top  fermentation  yeast  grows  and  forms  spores  readily 
at  14°  to  18°  C. ;  while  a  bottom  fermentation  yeast  grows  at  4°  to 
10°  C.  in  the  bottom  of  the  fluid,  and  forms  spores  only  with  difficulty. 

METHOD   OF   OBTAINING   PURE   CULTURES   ON   A 
SMALL  SCALE. 

1.  A  Pasteur  flask  containing  the  wort  to  be  experimented  with  is 
started  and  carried  on  as  vigorously  as  possible. 


244  THE  BLASTOMYCETES 

2.  To  this  growth  a  quantity  of  sterilized  water  is  added,  and  the 
yeast  cells  in  a  given  drop  counted  under  the  microscope. 

3.  Supposing   10  cells    to    be    present,  a   similar-sized  drop  is  now 
transferred  to  a  flask  containing  20  c.c.  of  water,  which  is  equivalent  to 
1  yeast  cell  for  each  2  c.c.  of  water. 

4.  The  flask  containing  the  20  c.c.  of  water  with  the  10  yeast  cells 
is  thoroughly  shaken,  and  this  liquid  divided  equally,  1  c.c.  being  placed 
in  each  of  twenty  flasks  containing  sterilized  wort. 

5.  If  the  separation  has  been  complete,  10  out  of  20  flasks  should 
contain  one  organism   each,   but   this  of  course  cannot    be   absolutely 
depended  on. 

6.  At  this  stage  Han  sen  shakes  the  flasks  very  vigorously  to  separate 
the  cells  as  much  as  possible,  and  places  the  flasks  in  the  incubator, 
allowing  them  to  remain  perfectly  still,  in  order  that  the  cells  may  sink 
to  the  bottom  or  become  attached  to  the  walls  of  the  flasks. 

7.  At  the  end  of  several  days  the  flask  is  carefully  lifted  and  exam- 
ined,  and  it  is  noted  whether  one   or  more  white  specks  have  been 
formed  on  the  walls  of  the  glass  ;  if  only  one  such  speck  is  found,  it  is  a 
pure  culture. 

This  method  is  especially  useful  when  the  yeast  plants  are  at  all 
weakly.  In  mixed  and  vigorous  species,  wort  gelatine  plate  cultures 
should  be  instituted.  For  the  methods  of  obtaining  pure  cultures  on  a 
large  scale,  see  the  works  of  Hansen  and  Jorgensen. 


SACCHAROMYCES  CEREVISI/E  I. 

This  is  known  as  the  Old  English  top-fermentation  yeast,  and  is 
used  by  brewers  and  bakers. 

Microscopical  Appearances. — Large  round  or  oval  cells,  11  to  4  /z, 
most  frequently  8  to  6  p  in  diameter.  These  cells  give  off  small  cells  by 
budding.  In  the  earlier  stages  of  film  formation  delicate  mycelial-like 
threads  are  formed,  which,  as  the  film  becomes  older,  grow  longer  and 
more  regular.  Nuclei  can  be  demonstrated  in  the  cells,  especially  in  old 
cultures,  when  stained  with  haemotoxilin,  haematine,  alum  solution,  or 
osmic  acid.  The  cells  are  sometimes  very  granular. 

Spore  Formation. — Ascospores  develop  after  twenty-four  hours  at  10° 
to  37°  C.,  but  most  rapidly  at  30°  C.,  most  slowly  (after  ten  days)  at  11°  to 
12°  C.,  and  below  this  temperature  the  formation  ceases.  For  the  develop- 
ment of  the  ascospores,  Hansen  employs  plaster-of- Paris  blocks,  which  are 
first  thoroughly  sterilized  by  heat.  A  small  portion  of  yeast  is  laid  on 
the  upper  surface,  and  the  lower  surface  set  in  a  small  vessel  containing 
water,  within  a  sterile  air  chamber,  the  whole  apparatus  being  placed  in 
an  incubator,  or  left  at  room  temperature.  Ascospores  can  also  be  grown 


FIG.  96.— Saccharomyces  Cerevisise  I.  and  Ascospores.    Cladius 
stain.     X  1000. 


[T.  Bowhill,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


SACCHAROMYCES  ELLIPSOIDEUS  245 

on  potatoes  prepared  according  to  Globig's  method.  (See  Technique, 
§78.) 

The  author  has  found  the  following  simple  arrangement  yield  most 
satisfactory  results.  An  oblique  plaster-of- Paris  block  is  prepared  so  that 
it  will  rest  in  the  bottom  of  an  ordinary  test-tube,  about  1  in.  in  diameter, 
containing  a  little  water  in  the  bottom,  which  is  plugged  and  sterilized. 
Some  yeast  is  placed  on  the  upper  portion  of  the  oblique  surface  of  the 
plaster  block,  when  the  tube  can  be  placed  in  the  incubator  or  left  at 
room  temperature. 

Staining  Reactions. — Dried  specimens  can  be  stained  with  fuchsin 
and  methylene  blue.  This  stain  is  also  used  to  differentiate  living  and 
dead  cells  in  hanging-drop  cultures,  the.  latter  alone  staining.  The 
author  has  found  that  ascospores  can  be  beautifully  demonstrated  by  the 
Cladius  method  of  staining,  §  12,  the  cells  being  stained  blue,  and  the 
spores  and  background  remaining  yellow  with  the  picric  acid  (see  Photo- 
micrograph, Fig.  96).  The  ascospores  can  also  be  stained  by  the  ordinary 
method  for  staining  spores  (see  §  26). 

Film  Formation. — This  takes  place  most  rapidly  (seven  to  ten  days) 
at  a  temperature  of  from  20°  to  22°  C.,  most  slowly  (two  to  three  months) 
at  6°  to  7°  C.,  and  ceases  altogether  above  38°  C.  and  below  5°  C. 

Biological  Characters. — On  Gelatine  Plates  it  forms  small  white 
colonies  ;  under  a  low  power  the  individual  yeast  cells  forming  the  surface 
colonies  can  be  observed. 

It  secretes  a  peculiar  substance  which,  acting  on  saccharose  or  crude 
cane  sugar,  inverts  it  to  invert  sugar.  This  latter  substance  is  fermented, 
a  similar  change  taking  place  in  dextrose  and  maltose,  alcohol  and  car- 
bonic acid  gas  being  formed,  accompanied  with  an  evolution  of  heat  and 
great  multiplication  of  the  yeast  cells.  On  lactose  or  milk  sugar  it  does 
not  seem  to  cause  any  change. 


SACCHAROMYCES    ELLIPSOIDEUS. 
(Divided  by  Hansen  into  I.  and  II.) 

SACCHAROMYCES  ELLIPSOIDEUS  I. 

This  is  a  '  wild '  species  of  wine  ferment.     It  is  found  on  the  sur- 
face of  fruit,  chiefly  on  wine  grapes. 

Microscopical  Appearances. — Round  or  oval  cells,  which  sometimes 
assume  a  sausage  form. 

Spore  Formation.— The  spores  are  from  2  to  4  ^  in  diameter,  two 
to  four  being  found  in  a  single  ascus.     They  are  developed  between  7J° 


246  THE  BLASTOMYCETES 

and  31  i°  C.,  most  rapidly  (in  twenty-one  hours)  at  25°  C. ;  above  32'5°  C. 
and  under  4°  C.  the  development  ceases. 

Film  Formation. — The  surface  membrane  is  formed  most  rapidly 
(eight  to  twelve  days)  at  33°  to  34°  C.,  most  slowly  (sixty  to  ninety  days) 
at  6°  to  7°  C.  It  is  always  a  delicate  membrane,  and  above  38°  C.  and 
under  5°  C.  development  ceases.  The  growth  is  most  characteristic 
between  13°  to  15°  C.,  when  it  consists  of  a  complicated  branching  mass 
of  elongated  cells  or  threads,  arranged  in  rows  with  lateral  processes 
coming  off  at  the  point  of  junction.  Secondary  branches  are  formed  at 
the  constrictions  of  the  primary  branches. 

On  Wort  Gelatine  the  colonies  present  a  net-like  appearance.  It 
causes  as  powerful  and  rapid  fermentation  as  the  Saccharomyces 
Cerevisiae  on  the  various  carbohydrates  on  which  that  ferment  acts. 

SACCHAROMYCES   ELLIPSOIDEUS   II. 

A  '  wild '  or  wine  fermentation  yeast,  which  gives  rise  to  the 
muddiness  of  beer. 

Microscopical  Appearances. — In  young  cultures  at  1 5°  C.  the  cells 
are  usually  somewhat  rounded  or  egg-shaped,  while  older  cultures 
exhibit  longer  mycelial  rods  with  forked  transverse  shoots  given  off  at 
the  joints. 

Spore  Formation. — The  spores  are  from  2  to  5  ^  in  diameter,  two 
to  four  being  found  in  a  single  ascus,  and  may  be  egg-shaped,  slightly 
irregular,  or  elongated.  They  are  developed  most  rapidly  at  29°  C., 
most  slowly  at  8°  C. ;  above  34°  C.  and  below  4°  C.  the  development 
ceases, 

Film  Formation. — It  is  essentially  a  low  yeast,  and  the  film  that 
forms  is  very  delicate.  At  33°  to  34°  C.  it  appears  in  three  to  four  days, 
but  not  for  five  to  six  months  at  3°  to  5°  C.  At  2°  and  at  40°  C.  no  film 
is  developed. 

SACCHAROMYCES   PASTORIANUS. 
Hansen  considers  this  an  impure  species,  and  divides  it  into  three. 

SACCHAROMYCES   PASTORIANUS   I.   (HANSEN). 

A  '  wild '  yeast,  the  spores  frequently  occurring  in  the  atmosphere 
of  breweries.  It  gives  an  unpleasant  bitter  taste  and  bad  smell 
to  beer. 

Microscopical  Appearances. — It  occurs  as  elongated  ellipsoidal 
or  pear-shaped  cells,  from  which  small  apical  or  lateral  branches  are 
sometimes  given  off. 


SACCHAROMYCES  PASTORIANUS  247 

Spore  Formation. — The  asci  are  usually  elongated  or  rounded, 
and  may  contain  two  spores  or  multiples  of  two  up  to  eight  or  even 
more,  which  vary  in  size  from  J  '5  up  to  5  fj..  They  are  developed  most 
rapidly  (seven  to  ten  days)  at  27 '5°  C.,  most  slowly  (fourteen  days)  at 
3°  to  4°  C.  The  development  ceases  at  '5°  C.  and  at  31°  C. 

Film  Formation. — The  films,  which  are  usually  very  delicate,  are 
developed  most  readily  (seven  to  ten  days)  at  from  26°  to  28°  C.,  most 
slowly  (five  to  six  months)  at  from  3°  to  5°  C. ;  development  ceases  at  34° 
and  2°  C.  Mycelial-like  threads  develop  freely  in  the  film  at  from  3°  to 
15°  C.,  and  most  irregular  forms  appear.  In  the  older  films  numerous 
irregular  club,  skittle-shaped,  and  other  forms  occur.  In  the  younger 
films  the  cells  are  usually  smaller  and  the  irregular  forms  less  frequent. 


SACCHAROMYCES    PASTORIANUS    II.    (HANSEN). 

This  was  also  separated  from  the  air  of  the  brewery.  It  is  a 
feeble  top  fermentation  yeast  when  growing  in  beer-wort.  It 
gives  rise  to  neither  cloudiness  nor  to  any  unpleasant  bitter  taste. 

Microscopical  Appearances. — The  sedimentary  cells  are  mostly 
elongated,  but  may  be  slightly  rounded,  varying  considerably  in  size. 
The  cells  found  in  the  film  are  rounded,  egg-shaped,  or  somewhat 
elongated. 

Spore  Formation. — The  asci  are  usually  elongated,  the  spores 
occurring  in  multiples  of  two  from  2  to  5  p,  in  diameter.  They  are 
developed  most  rapidly  (twenty-seven  hours)  at  23°  C.,  most  slowly 
(seventeen  days)  at  3°  to  4°  C.  ;  formation  ceasing  at  29°  C.  and  at  '5°  C. 
This  yeast  secretes  an  invertase  and  causes  fermentation  of  all  the 
carbohydrates  that  are  fermented  by  the  other  yeasts  of  this  group. 
In  old  cultures  of  the  films  the  cells  are  small,  thread-like,  and  very 
irregular  in  shape. 

SACCHAROMYCES  PASTORIANUS  III.  (HANSEN). 

According  to  Hansen  this  yeast  is  one  of  the  causes  of  turbidity 
in  beer. 

Microscopical  Appearances. — The  cells  are  very  similar  to  those 
of  the  sedimentary  yeast,  but  at  a  temperature  of  from  15°  down  to  3°  C. 
elongated  mycelial-like  threads  develop,  which  in  old  cultures  become 
still  more  characteristic.  In  the  Saccharomyces  Pastorianus  I.  the 
mycelial  threads  are  most  characteristic  at  13°  to  15°  C.,  while  at  15°  to 
3°  C.  the  cells  in  Saccharomyces  Pastorianus  II.  are  oval  and  rounded. 

Spore  Formation. — Similar  to  the  Saccharomyces  Pastorianus  II. 


248  THE  BLASTOMYCETES 

It  takes  place  most  rapidly  (twenty-eight  hours)  at  25°  C.,  most  slowly 
(nine  days)  at  8 '5°  C.,  and  ceases  at  29°  C.  and  at  4°  C. 

Film  Formation. — This  appears  in  the  form  of  small  flakes,  most 
rapidly  (seven  to  ten  days)  at  26°  to  28°  C.,  most  slowly  (five  to  six 
months)  at  3°  to  5°  C.,  and  ceases  altogether  at  34°  and  2°  C.  The  elon- 
gated or  sausage  form  cells  predominate,  but  large  and  small  round  and 
ovoid  cells  are  also  found  in  the  sediment  in  the  films  at  from  20°  to  28°  C. 

Biological  Characters. — In  cultures  grown  on  yeast-water  gelatine, 
at  the  end  of  sixteen  days  the  colonies  exhibit  peculiarly  fringed  edges. 
Grown  in  wort,  it  gives  rise  to  a  top  fermentation,  causing  considerable 
turbidity,  and  producing  alcohol  and  carbonic  acid  gas. 

SACCHAROMYCES  APICULATUS. 

found  in  fermented  wine  and  spontaneously  fermented  beer,  and 
in  hot  seasons  on  sweet  succulent  fruits — cherries,  plums,  grapes,  etc. 
In  winter  it  is  found  in  the  soil  beneath  the  trees  that  bear  these 
summer  fruits. 

Microscopical  Appearances. — In  cultivation  fluids  the  cells  are 
lemon-shaped,  the  buds  are  lemon-shaped,  and  in  older  cultures  oval. 

Spore  Formation  absent.  Hence  it  cannot  be  classified  as  a  true 
yeast. 

Biological  Characters. — When  dried  in  a  thin  layer  it  is  killed, 
which  accounts  for  it  not  developing  on  unripe  fruit.  It  is  a  bottom  fer- 
mentation yeast,  causing  feeble  alcoholic  fermentation.  It  does  not  invert 
cane  sugar,  but  acts  on  dextrose  in  yeast  water,  the  fermentation  being 
incomplete.  Mixed  with  Saccharomyces  it  retards  the  action  of  the 
latter. 

SACCHAROMYCES   ANOMALUS. 

Found  in  brewery  yeast. 

Microscopical  Appearances. — Small  oval  cells. 

Spore  Formation  present,  in  the  form  of  hemispheres  with 
projecting  rims,  whereby  they  are  shaped  like  a  hat;  the  optimum 
temperature  for  their  development  is  25°  C. 

SACCHAROMYCES   MARXIANUS. 

First  found  in  wine. 

Microscopical  Appearances. — Small  ellipsoidal  and  egg-shaped 
cells,  with  sausage-shaped  cells  here  and  there ;  often  arranged  in 
colonies. 


SACCHAROMYCES  MEMBRANE  FACIENS  249 

Spore  Formation.  —  Spores  are  not  freely  developed ;  on  solid 
media  they  are  more  frequent,  being  usually  oval  or  kidney-shaped. 

Film  Formation. — The  film  develops  very  slowly,  and  consists  of 
oval  and  short  sausage-shaped  cells. 

Biological  Characters. — In  beer-wort  it  is  not  very  active.  It 
does  not  ferment  maltose,  but  acts  strongly  on  saccharose,  which  it 
inverts  and  then  ferments  with  great  rapidity.  It  also  acts  upon 
dextrose. 

SACCHAROMYCES   MEMBRAN.EFACIENS. 

Forms  a  bright  yellow,  tough  scum  on  beer- wort,  composed  of  long 
and  sausage-like  cells,  occurring  either  singly  or  closely  packed 
together. 

Spore  Formation. — Spores  are  formed  rapidly. 

Biological  Characters. — Nutrient  gelatine  is  liquefied.  It  does 
not  cause  any  fermentation  of  ordinary  carbohydrates  and  does  not 
invert  cane  sugar. 

SACCHAROMYCES    EXIGUUS. 

Found  in  German  yeast  by  Hansen.  It  forms  no  mycelial  threads 
on  beer- wort  or  on  solid  media.  It  forms  spores  but  sparsely.  The 
film  is  very  delicate,  and  consists  of  short  rod-shaped  or  ovoid  cells. 
It  causes  the  same  changes  in  sugars  as  the  Saccharomyces  marxianus. 


SACCHAROMYCES   ACIDI   LACTICI. 

Occurs  as  ellipsoidal  cells. 

On  Gelatine  Plates  and  Agar  Surface  Cultures  it  forms  white,  glistening 
colonies. 

In  Gelatine  Slab  Cultures,  club-shaped  outgrowths  are  given  off  from 
the  inoculation  track. 

On  Potatoes  it  forms  a  brownish  coating. 

Milk  is  coagulated,  an  acid  being  formed.  In  solutions  of  milk  sugar 
it  forms  alcohol. 

MYCODERMA   CEREVISLE   ET   VINI. 

Like  all  other  forms  of  mycoderma  it  is  distinguished  from  the 
saccharomyces  by  forming  no  spores  (ascospores). 


250  THE  BLASTOMYCETES 

Microscopical  Appearances. — Long  cells,  which  do  not  transmit 
light  so  strongly  as  the  saccharomycetes. 

Biological  Characters. — On  wort  gelatine,  small  dim  light  grey 
spots,  which  either  spread  over  the  surface  or  cause  a  shell-shaped  cavity 
in  the  medium. 

On  Wort  a  greyish-white,  thickly  matted  film  develops,  the  optimum 
temperature  being  15°  C. 

Actions. — It  causes  only  a  weak  alcoholic  fermentation,  no  acetic  acid 
fermentation,  but  exerts  a  deleterious  action  on  fermented  fluids  by 
producing  abnormal  chemical  changes. 

THE   TORUL.E. 

These  yeasts  are  widely  distributed  in  nature,  and  occur  in  round  or 
elongated  forms  and  develop  no  spores.  Reproduction  takes  place  by 
budding,  and  often  at  the  same  time  a  mycelium  is  formed.  Gelatine 
media  are  not  liquefied.  The  various  forms  are  distinguished  by  the 
colour  of  the  pigment  produced. 

1.  White,  Hefe. 

2.  Rose, 

3.  Black,       „ 

They  cause  only  a  very  weak  alcoholic  fermentation.  There  are, 
however,  some  species  that  grow  with  distinct  fermentation  properties. 


PATHOGENIC    BLASTOMYCETES. 
SACCHAROMYCES   HOMINIS. 

Found  in  an  infectious  disease,  which  began  with  a  subperiosteal 
inflammation  of  the  tibia,  terminating  in  a  chronic  pyaemia. 

Microscopical  Appearances. — Round  or  oval  cells,  double  con- 
toured, and  possessing  capsules. 

Biological  Characters. — On  Gelatine  Plates,  prominent,  round, 
non-liquefying  colonies. 

On  Agar,  a  white  coating. 

On  Potatoes,  a  greyish-brown  coating. 

On  Blood  Serum,  a  dim  drop-like  coating. 

Bouillon  is  clouded,  a  film  growing  on  the  surface. 

In  Grape  Sugar  Bouillon  fermentation  takes  place,  alcohol  and  CO2 
being  formed. 

Pathogenesis.— In  rabbits  a  local  abscess  forms.  Mice  die  from 
septic  changes. 


PATHOGENIC  BLASTOMYCETES  251 


SACCHAROMYCES   LITOGENES. 

Found  in  the  lymph-glands  of  an  ox  affected  with  carcinoma  of 
the  liver. 

Microscopical  Appearances. — Large  and  small  round  cells  with  a 
membrane. 

Biological  Characters.  —  The  growth  in  the  various  media  is 
similar  to  the  Saccharomyces  neoformans,  except  on  potatoes,  when  an 
intense  brown  coating  develops. 

Pathogenesis — In  guinea-pigs  a  tumour  forms  at  the  point  of 
inoculation  and  nodules  in  the  organs.  The  yeast  cells  in  the  centre  of 
the  nodules  very  frequently  degenerate. 

SACCHAROMYCES  NEOFORMANS. 

Found  in  the  juice  of  fruits. 

Microscopical  Appearances. — Round  or  elliptical  cells  with  re- 
f  ractile  granules  and  a  double  contour,  which  increase  by  budding. 

Biological]  Characters.  —  On  Gelatine  Plates,  round,  cup-shaped, 
non-liquefying  colonies. 

In  Gelatine  Stab  Cultures,  a  granular  growth  along  the  inoculation 
track. 

On  Agar,  a  dry  film  develops. 

On  Potatoes,  a  white  elevated  growth. 

Milk  is  not  coagulated. 

In  Sugar  Bouillon  a  sediment  forms,  and  often  a  film  is  developed. 

Pathogenesis. — In  guinea-pigs  a  tumour  forms  at  the  point  of 
inoculation,  and  nodules  develop  in  the  internal  organs. 

SACCHAROMYCES   SUBCUTANEUS   TUMEFACIENS. 

Found  in  a  myxomatous  tumour  of  the  upper  part  of  the  thigh. 

Microscopical  Appearances. — Oval  or  round  cells,  frequently 
possessing  large  transparent  capsules.  The  cells  increase  by  budding. 

Biological    Characters.  —  In  Gelatine  Stab   Cultures   the   develop- 
ment occurs  in  small  colonies  ;  it  causes  no  liquefaction  of  the  medium. 
On  Agar,  a  thick  creamy  growth. 

On  Potatoes,  an  extensive  white  coating,  which  later  becomes  brown. 
On  Acid  Beer-wort  Agar,  a  brown  coating. 
In  Alkaline  Bouillon,  a  slight  sediment. 
In  Beer-wort,  a  thick  sediment  without  film  formation  on  the  surface. 


252  THE  BLASTOMYCETES 

It  causes  a  slight  fermentation  of  saccharose.  It  forms  ethylic 
alcohol  and  acetic  acid. 

Pathogenesis. — White  mice  and  rats  are  susceptible,  extensive 
local  vegetations  being  produced.  Microscopically  examined,  the 
tumours  exhibit  no  definite  structure,  but  appear  to  consist  of  an 
extensive  parasitic  infiltration. 

HYDROPHOBIA. 

Memmo,  of  the  Hygienic  Institute,  University  of  Rome,  has  recently 
stated  in  a  contribution  regarding  the  etiology  of  hydrophobia,  that  in 
some  cases  he  found  a  blastomycete  possessing  pathogenic  properties 
which  he  considers  the  cause  of  the  disease.  The  fungus  was  found  in 
the  cerebro-spinal  fluid,  the  substance  of  the  brain  aqueous  humour, 
stroma  of  the  parotid  gland,  and  in  the  saliva,  but  never  in  the  heart's 
blood  or  other  organs.  Liquid  culture  media  were  used,  especially 
bouillon  containing  tartaric  acid,  and  glucose  of  weak  acid  reaction. 
The  cultures  did  not  begin  to  develop  for  ten  days.  In  material  from 
ten  healthy  dogs  and  twenty  rabbits,  no  similar  cultures  were  produced. 
Culture  fluids  exposed  to  the  air,  and  mixed  with  dust  found  in  the 
laboratory,  yielded  no  growth  of  the  above  fungus. 

With  cultures  of  three  or  four  generations  from  four  different  out- 
breaks of  hydrophobia,  the  purity  of  some  being  tested  by  plate 
cultures,  rabbits,  guinea-pigs,  and  dogs  were  inoculated,  mostly  sub- 
cutaneously,  but  also  by  iiitraperitoneal  and  intradural  methods.  In 
eleven  to  twenty  days  some  of  the  guinea-pigs  and  rabbits  were  affected 
with  paralysis  of  the  hind  extremities,  followed  shortly  by  death. 

In  thirty  to  sixty  days  the  dogs  began  to  emaciate,  became  rabid, 
vomited,  and  finally  paralysis  of  the  extremities  preceded  death.  The 
brain  substance  of  the  infected  animals  was  infectious  for  other  animals. 
The  fungus  has  also  been  found  in  sections  of  the  spinal  cord  of  a  boy 
dead  of  hydrophobia,  stained  by  Sanfelice's  method. 


PART    VI. 

THE    PROTOZOA,   OR  ANIMAL   PARASITES. 

AMGEBA    COLI. 

Found  in  the  human  intestines,  in  the  stools,  in  dysentery,  and 
in  the  diarrhoea  in  abscess  of  the  liver. 

Microscopical  Appearances. — During  the  resting  stage  they 
appear  as  almost  homogeneous  cells,  while  during  motility  a  slightly 
refractive  ectoplasm  and  strongly  refractive  entoplasm  with  a  granular 
appearance  can  be  distinguished.  Vacuoles  are  also  frequently  present 
in  the  entoplasm,  as  well  as  foreign  bodies,  red  blood-corpuscles,  and 
bacteria.  The  large  nucleus,  with  a  nucleolus,  is  plainly  visible  in  dead 
forms,  stained  specimens,  or  by  the  addition  of  acetic  acid. 

Motility. — This  is  due  to  the  presence  of  pseudopodia,  in  which 
rounded  blunt  continuations  of  the  ectoplasm  extend  into  the  entoplasm. 

The  Amoeba  coli  multiply  by  dividing  in  two.  All  attempts  at 
cultivation  have  up  to  the  present  failed.  The  Amoeba  coli  found  in  the 
normal  intestinal  contents  cannot  be  distinguished  morphologically  from 
that  found  in  dysentery,  but  the  latter,  when  introduced  into  the  rectum 
of  cats,  causes  an  ulcerative  haemorrhagic  inflammation  of  the  large 
intestines.  Furthermore,  the  dysentery  amoeba  are  genuine  tissue 
parasites,  penetrating  deep  into  the  submucous,  sometimes  as  far  as  the 
serous  coating  of  the  intestines. 

Method  of  Examining  Stools  for  Bacteria. — The  stool  for  exa- 
mination should  be  quite  recent,  and  should,  if  possible,  be  received 
in  a  vessel  previously  warmed  to  body-heat.  In  a  watery  stool  a  drop  is 
taken,  preferably  from  the  red  jelly-like  part  showing  bloody  mucus,  and 
examined  on  a  cover-glass.  If  more  solid  but  unformed,  the  stool  is 
diluted  with  warm  normal  saline  solution,  and  treated  as  above. 

In  solid  formed  stools  some  of  the  mucous  slime  attached  is  examined. 
In  making  a  permanent  preparation  some  of  the  material  is  smeared 
very  thinly  on  the  cover-glass,  and  before  there  is  time  for  drying  it  is 
passed  into  absolute  alcohol  to  fix.  Their  staining  reactions  are  not  as 
pronounced  as  those  of  the  bacteria,  and  methylene  blue  gives  the  best 


254  THE  PROTOZOA 

results,  and  we  get  the  nucleus  and  the  protoplasm  both  stained,  but  the 
former  more  deeply.  Sections  stain  with  cosine  and  hsematoxylin  as  well 
as  methylene  blue. 

PARAM^ICIUM   COLL 

Found  in  the  intestines  of  man  and  swine. 

Microscopical  Appearances. — Elliptical  infusoria,  their  whole 
surface  being  covered  with  cilia.  In  man  they  are  60  to  70  /u  long,  and 
in  swine  70  to  100  /x  long.  The  mouth  is  funnel-shaped,  the  nucleus 
bean-shaped,  and  they  possess  two  contractile  vacuoles.  They  multiply 
by  dividing  into  four  and  by  conjugation,  also  sometimes  becoming 
encysted. 

COCCIDIA. 

The  best  known  coccidia  are  those  found  in  the  rabbit  and  the  fowl, 
and,  according  to  Rieck,  there  are  two  different  varieties — a  liver  and  a 
bowel  species,  the  former  causing  peculiar  white  or  abscess-like  cavities 
in  the  liver  (due  to  local  dilatation  of  the  bile-ducts),  the  other  inhabiting 
the  intestines  and  causing  a  most  acute  and  fatal  inflammation. 
M'Fadyean  records  an  outbreak  amongst  pheasants  of  the  intestinal 
variety,  where  the  coccidia  penetrated  the  glands  of  Lieberkiihn  and 
invaded  their  epithelium.  The  same  author  also  mentions  an  outbreak 
amongst  lambs. 

Zschokke,  Hess,  and  Guillebeau  mention  a  form  of  dysentery 
amongst  cattle  in  Switzerland,  produced  by  coccidia,  and  known  as  fred 
dysentery  '  and  '  dysenteria  haemorrhagica  coccidiosa.'  It  has  also  been 
produced  experimentally  in  cattle  with  sporulating  coccidia. 

The  coccidia  are  unicellular  animal  parasites  belonging  to  the  class 
Sporozoa.  In  the  adult  state  they  are  ovoid  in  shape  and  enclosed  in  a 
double-contoured  shell  or  cyst,  flattened  at  one  of  its  poles,  30  to  50  n 
long  and  14  to  28  /x  broad.  The  protoplasmic  contents  of  the  encysted 
forms  separate  from  the  wall  and  contract  into  a  ball-shaped  mass. 


COCCIDIUM   OVIFORME. 

This  parasite  is  found  in  the  rabbit's  liver,  and  sometimes  in  the 
intestinal  canal.  It  enters  the  gall-ducts  by  way  of  the  ductu, 
choleduchus,  thence  into  the  epithelial  cells,  and,  increasing  in  size, 
becomes  encysted.  It  is  surrounded  by  a  slender  external  membrane 
and  a  shining  double  inner  membrane.  The  permanent  cysts  have 
granular  contents  and  a  nucleus.  The  development  does  not  proceed 


FIG.  97.—  Coccidium  Oviforme.    Cover-glass  specimen  from  liver  of 
rabbit.    Stained  by  Ehrlich's  method.     X  750. 


M/-  <f":'t<Bsrv 


;^ 

-/•^ 


FIG.  98.— Section  of  a  rabbit's  liver,  showing  Coccidia.     Unstained. 
X  300. 


[T.  Bowhill,  F.R.C.V.S.,  Photo.,  Edinburgh, 


COCCIDIUM  OV1FORME  255 

further  in  the  rabbit's  liver,  but  outside  the  animal's  body,  and  in  contact 
with  moisture,  the  plasma  of  the  cell  divides  into  four  oval  mother  spores, 
each  of  which  again  divides  into  two  sickle-formed  daughter  spores. 
When  these  sickle-formed  spores  gain  entrance  to  an  animal's  stomach, 
the  membrane  of  the  mother  spores  is  dissolved  by  the  gastric  juice,  the 
free  spores  entering  the  intestines  and  ultimately  the  gall-ducts.  All 
the  parasites  in  an  affected  liver  do  not  complete  the  above  cycle  of 
development,  but  sporulate  during  their  growth  in  the  epithelial  cells, 
exhibiting  4  to  50  sickle-formed  nucleus-containing  bodies,  which  finally 
cause  a  general  disease  of  the  liver.  White  particles  are  sometimes 
found  floating  in  the  bile,  consisting  of  masses  of  coccidia,  and,  according 
to  Rivolta,  the  parasite  sometimes  develops  in  the  epithelium  of  the 
gall-bladder. 

Staining  Reactions. — The  parasite  stains  well  with  haematoxylin 
and  eosin  in  sections  and  cover-glass  specimens.  The  author  obtained 
some  specimens  from  a  Jack-rabbit's  liver  in  California  in  1895,  which 
exhibited  the  same  reactions  towards  Ehrlich's  aniline  water  fuchsin  as 
the  bacillus  of  tuberculosis.  (See  Photomicrograph  of  same,  Fig.  97  ; 
for  Photomicrograph  of  section  of  the  same  liver,  showing  the  Coccidia 
unstained,  see  Fig.  98.) 

According  to  Leuckart,  the  intestinal  species  is  known  as  Coccidium 
perforans,  but  that  it  is  a  distinct  species  is  not  exactly  proven,  as  both 
forms  frequently  coexist  in  the  same  host,  the  difference  probably  arising 
from  the  different  positions  of  the  parasites  in  the  affected  animals. 

In  man,  cases  of  coccidia  are  recorded  in  the  liver,  kidneys,  and 
pleural  exudate. 

Rivolta  also  mentions  the  presence,  along  with  the  Coccidium 
oviforme  in  the  rabbit's  liver,  of  other  coccidia  belonging  to  the  species 
Elimeria  falciformis. 


PLASMODIUM   MALARLE. 

This  parasite  was  discovered  in  the  blood  of  malarial  patients  by 
Laveran  in  1882. 

Microscopical  Appearances. — The  young  forms  of  the  parasite 
are  about  1  to  2  /i  in  size,  colourless  and  motile  ;  in  stained  specimens 
small  nuclei  are  visible.  The  parasites  either  cling  to  the  outside  of 
the  blood-corpuscles,  or  enter  the  interior  of  the  same.  The  parasites 
developing  in  the  interior  of  the  red  blood-corpuscles,  form  at  the 
expense  of  the  haemoglobin,  or  brown  or  black  granular  pigment.  At 
the  height  of  its  growth  the  parasite  may  fill  the  whole  blood-cell ;  and 
in  this  condition,  when  it  sporulates,  the  spores  rupture  the  remains  of 
the  blood  discs,  thus  liberating  themselves. 


256  THE  PROTOZOA 


VARIOUS  TYPES  OF  MALARIAL  PARASITES. 

(1.)  The  Quartan  Parasite. — The  cause  of  quartan  malaria  has  an 
incubation  period  of  seventy-two  hours  ;  they  are  not  strongly  motile. 
At  first  the  parasite  occupies  J  or  J  of  the  blood-cell,  and  large  quanti- 
ties of  granular  pigment  are  present.  Losing  its  motility  it  increases 
in  size,  until  it  appears  to  fill  the  whole  blood-cell.  It  then  forms 
spores,  arranged  like  the  ray-flowers  in  a  daisy-marguerite  form,  having 
eight  to  twelve  round  spores,  the  whole  period  of  development  occupy- 
ing about  three  or  four  hours.  The  segmentation  takes  place  both 
before  and  during  the  febrile  stage ;  and  about  three  hours  before  the 
outbreak  of  the  rigors,  the  first  mature  spores  are  visible  in  the  blood. 

The  red  blood-corpuscles  affected  with  the  quartan  parasite  do  not 
alter  their  size.  The  parasites  sometimes  sporulate  before  completely 
filling  the  blood-cell,  when  they  only  form  four  to  five  spores.  Cilia 
are  only  observed  in  young  forms.  If  many  generations  of  the  quartan 
parasite  are  present  in  the  blood,  then  it  causes  quartan  duplex  or 
triplex,  or  an  irregular  type  of  fever. 

(2.)  The  Tertian  Parasite. — The  cause  of  the  tertiana  form  of  malaria 
develops  in  forty-eight  hours ;  the  young  form  resembles  the  quartan 
parasite.  It  is  strongly  motile,  possessing  pseudopodia,  contains  pig- 
ment, enlarges  the  red  blood-corpuscles  containing  them,  and  sporulates 
in  the  form  of  a  rosette,  or  like  a  sunflower  containing  fifteen  to  twenty 
spores  which  are  smaller  than  those  of  the  quartan  parasite,  the  nucleus 
only  being  observed  with  difficulty.  The  free  spores  infect  fresh  blood- 
corpuscles  in  a  short  time,  completing  the  same  above-mentioned  cycle  of 
development.  All  the  tertian  parasites  do  not  sporulate,  a  large  number 
remaining  sterile.  These  sterile  parasites  are  as  large  or  larger  than 
the  sporulating  forms,  and  the  pigment  remains  motile.  (Laveran  con- 
siders this  a  degenerative  process.)  They  can  be  observed  in  the  blood 
hours  after  the  attack,  and  also  during  the  fever-free  days.  The 
process  of  sporulation  is  coincident  with  the  fever  paroxysms  in  the 
tertiana  form.  Golgi  found  that  three  hours  before  the  rigors  com- 
menced, the  temperature  had  already  begun  to  rise,  and  that  the  first 
spores  were  already  apparent  in  the  blood,  but  they  were  most  plentiful 
during  the  rigors.  The  full  -  grown  tertian  parasite  often  possesses 
flagella.  (Kruse  considers  this  condition  likewise  a  degeneration.)  The 
tertian  parasites  cause  typical  tertiana  ;  two  generations  of  the  same  can 
produce  a  false  quotidiana.  Tertiana  duplex  generates  several  generations 
separated  about  twenty-four  hours  from  each  other,  causing  irregular 
fever. 

(3.)  The  Quotidian  Parasite. — The  commencement  of  the  cycle  of 
development  of  this  parasite,  which  requires  twenty-four  hours  for  its 
completion,  is  similar  to  that  of  the  previous  forms.  The  young  form 


MALARIA  257 

consists  of  plasma  and  a  nucleus,  is  devoid  of  pigment,  and  infects  the 
red  blood-corpuscles.  The  parasite  is  strongly  motile,  which  assists  in 
its  identification,  as  on  account  of  its  fine  contour  and  colour,  which  is 
only  a  little  paler  than  the  blood-corpuscle,  it  is  scarcely  distinguishable 
from  the  latter.  The  parasite  loses  its  motility  in  blood  preparations 
quickly,  one  hour  at  the  latest.  A  whitish  ring  is  now  formed,  and 
owing  to  an  outgrowth  at  one  part  of  the  periphery,  it  frequently 
resembles  a  signet  ring. 

Mannaberg  considers  these  forms  do  not  live  within  the  blood  cell, 
but  adhere  firmly  to  the  outside.  The  red  spot  in  the  centre,  according 
to  the  same  authority,  is  due  to  thinning  of  the  plasma,  and  the  shining 
through  of  the  underlying  red  blood-corpuscle.  The  ring-form  can 
again  return  to  an  amoeboid  condition.  The  amoeboid  parasite  does 
not  grow  very  large,  occupying  at  the  most  about  one-third  of  the  red 
blood-corpuscle,  forming  a  very  fine  pigment  on  the  periphery,  and 
exhibits  slight  motility.  In  twenty  hours  the  pigment  concentrates  in 
the  middle  or  edges  of  the  cell  in  dark  dormant  lumps,  and  the  parasite 
divides  inside  the  red  blood-corpuscle  into  small  spores  (five  to  ten). 

According  to  Marchiafava  and  Celli,  the  sporulation  takes  places 
only  in  the  internal  organs  of  the  body,  almost  not  at  all  in  the  peri- 
pheral blood.  The  spores  are  plentiful  in  blood  taken  from  the  spleen, 
while  in  blood  from  the  finger  they  are  either  absent,  or  only  present  in 
very  scanty  numbers.  Red  blood-corpuscles  affected  with  the  quotidian 
parasites  shrink,  and  acquire  a  yellow  copper-colour,  and  when  the  para- 
site is  several  days  in  the  blood,  the  Laveran  half-moon  or  disc-shaped 
bodies,  and  additional  forms  (spindle  or  cigar-shaped,  and'  spherical) 
appear.  The  quotidian  parasite  causes  quotidiana,  and  when  several 
generations  are  in  evidence,  a  continual  or  irregular  fever.  The  fever  caused 
by  the  tertian  and  quartan  parasites  is  distinguished  from  that  of  the 
quotidian  parasite  by  the  malignant  clinical  symptoms  of  the  latter — 
obstinate  recurrence,  great  anaemia,  and  other  pernicious  symptoms  (diar- 
rhoea, cachexia,  coma,  etc.),  the  relapse  appearing  about  fourteen  to  fifteen 
hours  after  the  first  cycle.  The  half-moon  forms  are  considered  answer- 
able for  the  recurrent  form.  They  are  found  in  the  blood  during  the 
fever-free  stages,  and  by  segmentation  or  genuine  spore  formation  can  give 
rise  to  the  formation  of  new  amoeboid  forms.  Then  the  new  paroxysms 
do  not  exhibit  a  genuine  recurrence,  but,  according  to  Golgi,  the  mani- 
festation of  a  long-interval  type.  According  to  other  authorities  this  is 
disputed,  and  the  half-moon  variety  is  considered  a  degenerative  form 
incapable  of  forming  new  individuals. 

(4.)  The  Malignant  Tertian  Parasite  is  considered  by  Marchiafava  and 
Begnami  as  a  distinct  species  closely  resembling  the  quotidian  parasite, 
but  distinguished  from  the  latter  by  completing  its  cycle  of  development 


258  THE  PROTOZOA 

in  forty-eight  hours,  being  larger,  and  at  the  time  of  sporulation  occupies 
one-half  to  two-thirds  of  the  blood-corpuscle;  no  pigment  is  formed  for 
over  twenty-four  hours,  and  when  it  is  formed  the  motility  of  the 
parasites  is  not  impaired.  Circular  forms,  and  later  on  half-moon  forms, 
also  develop.  It  has  usually  8  to  15  spores.  The  infected  blood- 
corpuscles  become  copper-coloured,  shrivel  easily,  but  never  hypertrophy. 
These  peculiarities  distinguish  this  parasite  from  Golgi's  ordinary  tertian 
parasite,  which,  moreover,  in  all  described  stages  is  larger,  richer  in 
pigment,  and  forms  more  spores,  15  to  20.  The  malignant  tertian 
parasite  generates  a  severe  tertian  form  of  fever,  the  chart  showing 
peculiar  curves,  with  very  short  fever-free  intervals,  often  only  of  a  few 
hours'  duration,  and  with  regular  pseudo-crises  ending  in  continued  or 
irregular  fever.  The  quotidian  parasite  also  exhibits  malignant 
characters. 

Mixed  Infection. — In  many  cases  of  intermittent  fever  various  forms  of 
the  above-mentioned  parasites  are  present  at  the  same  time  in  the 
blood.  Quotidian  parasites  frequently  occur  beside  non-pigmented,  and 
the  quartan  sometimes  beside  the  tertian  parasite.  Golgi  found  in  one 
case  three  generations  of  the  quartan  parasite  and  two  of  the  tertian 
parasite.  In  the  fever  curve  of  the  chart  we  may  find  the  different 
parasites  showing  their  effects  simultaneously,  or  an  irregular  type  of 
fever  may  be  manifested.  It  also  happens  that  during  the  course  of  the 
fever  only  one  form  of  parasite  causes  the  changes,  the  others  apparently 
having  no  influence. 

According  to  Lekowicz's  investigations  of  malignant  and  ordinary 
(tertiana  and  quartana)  malarial  fever,  the  parasites  of  both  groups 
develop  in  a  similar  manner,  but  are  differentiated  as  follows  : — (a)  Length 
of  the  period  of  incubation ;  (6)  the  size  and  form  of  the  parasite  found 
in  the  same  stage  of  development ;  (c)  the  presence  of  melanin ;  (d)  the 
presence  of  flagella  and  the  number  of  spores  present.  However,  the 
greatest  differentiation  is  that  the  parasites  of  ordinary  malaria  develop 
within  the  cell  (endoglobular),  while  those  of  the  malignant  forms  are 
developed  without  the  cell  (extraglobular).  The  former  grows  within  the 
red  blood-corpuscles,  the  latter  rolling  the  red  corpuscles  around  them- 
selves like  a  caterpillar  does  a  leaf,  whereby  a  part  of  their  surface 
remains  free,  enabling  them  to  attach  themselves  to  the  wall  of  the  blood- 
vessels. When  the  parasite  is  separated  from  the  blood-corpuscles  by 
pressure  of  the  cover-glass  in  cases  of  ordinary  malaria  forms,  the  wall 
of  the  corpuscle  is  ruptured,  and  the  pigment  escapes  outwards,  the 
remainder  of  the  corpuscle  being  immediately  decolorized.  In  cases  of 
malignant  malaria  the  wall  and  the  pigment  of  the  blood-corpuscle 
remain  intact,  and  the  parasite  is  not  in  the  interior  of  the  cell,  but  lies 
in  a  small  cavity  formed  by  the  rolling  process.  The  growth  of  the 
parasites  in  both  instances  is  identical.  At  the  end  of  the  febrile  stage 


MALARIA  259 

small  protoplasmic  bodies  appear,  possessing  amoeboid  movement,  which 
at  rest  assume  a  ring  form,  finally  either  penetrating  the  red  blood- 
corpuscles,  or  rolling  the  corpuscles  around  themselves  and  absorbing 
the  haemoglobin.  The  duration  of  the  vegetative  period  of  the  parasite 
causing  the  malignant  types  is  disproportionately  longer  than  by  the 
parasites  of  tertiana  and  quartana.  The  fully-developed  parasite  is  about 
the  size  of  a  red  blood-corpuscle,  and  in  ordinary  malarial  fever  is 
spherical,  and  in  malignant  malaria  half-moon  or  disc-shaped  ;  from  these 
forms  further  oval  and  spherical  forms  also  originate.  At  the  height 
of  this  stage  the  parasites  appear  (probably  owing  to  the  lower  tempera- 
ture) to  possess  flagella,  the  tendency  thereto  varying  with  each  group. 
The  protozoa  of  the  malignant  forms  develop  a  distinct  double-contoured 
cuticle,  while  those  of  ordinary  malarial  fever  possess  an  outer  fine  mem- 
brane ;  nevertheless,  spore  formation,  excapsulation,  and  dissemination  of 
the  young  spores  is  analogous  in  both  forms.  In  the  sporulating  bodies 
of  the  half-moon  variety,  Lekowicz  has  counted  as  many  as  thirty  spores. 
He  also  distinguishes,  as  characteristic  of  the  group,  an  extraglobular 
incubative  period  of  more  than  three  days'  duration  ;  and  of  the  half-moon 
or  disc-shape  of  the  fully  developed  individuals,  four  forms  are  determined, 
namely — 

1.  Haemosporidium  undecimanae.     (Period  of  incubation,  ten  days.) 

2.  Haemosporidium  sedecimanae  (fifteen  days). 

3.  Haemosporidium  vigesimo-tertianae  (twenty-two  days). 

4.  Haemosporidium  (?)  (incubative  period  unknown). 

The  fever  paroxysm  coincides  with  the  sporulation  of  a  generation  of 
these  parasites.  In  the  malignant  forms  a  quotidian  and  tertian  type  is 
frequently  observed,  because  many  generations  of  parasites,  differing  in 
age  from  twenty-four  to  twenty-eight  hours,  are  present.  The  presence 
of  the  tertian  type  in  these  cases  is  explained  by  a  severe  attack  inter- 
fering with  the  formation  of  the  active  living  spores  of  the  immediately 
following  twenty-four  hours'  younger  generation,  without  influencing  the 
forty-eight  hours'  younger  generation,  in  which  encapsulation  has  not  yet 
taken  place.  The  half-moon  forms  are  by  no  means  inaccessible  to  the 
action  of  quinine  as  heretofore  accepted.  Their  resistance  is  only  so 
altered  that  the  quinine  impairs  the  vitality  of,  or  destroys,  the  complete 
half-moon  forms  capable  of  forming  spores  of  several  generations  of 
simultaneous  existing  parasites,  but  it  does  not  prevent  previously  existing 
young  and  half-grown  sporidia  in  the  internal  organs  developing  into 
half-moon  forms.  After  treatment  with  quinine  the  half-moon  forms  are 
still  present  one  to  two  weeks  later  in  the  blood. 

METHOD  OF  PREPARING  DRY  SPECIMENS. 

1.  The  cover-glass  with  the  drop  of  blood  in  its  centre  is  drawn 
quickly  across  another  cover-glass,  and  both  are  azr-dried,  protected  from 
dust. 


260  THE  PROTOZOA 

2.  Fix  five  to  thirty  minutes  in  a  mixture  of  equal  parts  of  absolute 
alcohol  and  ether. 

3.  Dry  between  two  pieces  of  filter  paper. 

4.  Stain  in  a  watery 'solution  of  methylene  blue ;  wash  with  water. 

5.  Contrast  stain  in  a  2  per  cent,  solution  of  eosin  in  60  per  cent, 
alcohol. 

PLEHN'S  METHOD. 

Stain. 

Methylene  blue  concentrated  watery  solution,  .  .  60 
J  per  cent,  eosin  solution  in  75  per  cent,  alcohol,  .  20 
Aqua  distillata,  ........  40 

1.  Place  the  specimen  in  the  stain  five  to  ten  minutes. 

2.  Wash  in  water  and  dry. 

3.  Mount  in  xylol  balsam. 

Results.- — The  cells  containing  haemoglobin  are  stained  red,  the  plasma 
and  body  of  the  parasite  a  more  or  less  intense  blue. 

Many  other  staining  and  fixing  methods  are  used,  but  examination  of 
the  fresh  specimens  and  staining  by  the  above-mentioned  methods  will 
be  found  sufficient  for  diagnostic  purposes.  To  observe  the  nucleus 
substance,  the  ordinary  methylene  blue  method  is  sometimes  sufficient,  or 
the  specimen  is  fixed  in  a  mixture  of  acetic  and  picric  acids  and  stained 
with  haemotoxylin,  or  examined  in  fresh  blood  to  which  some  methylene 
blue  or  fuchsin  is  added. 

Mode  of  Infection. — The  cultivation  of  the  parasite  of  malaria  has 
not  yet  been  accomplished,  and  infection  of  animals  has  also  not 
succeeded.  The  parasite  is  only  known  as  a  parasite  of  the  blood  of 
man,  and  of  where  and  in  what  manner  they  exist  outside  the  body 
nothing  is  known,  therefore  the  knowledge  of  the  mode  of  infection  is 
very  limited.  Malaria  can  be  communicated  from  man  to  man  by  means 
of  the  blood.  Gerhardt  did  it  subcutaneously,  and  later  it  was  accom- 
plished both  by  subcutaneous  and  intravenous  injections  of  the  blood  of 
malarial  patients.  Nevertheless,  malaria  is  not  a  contagious  disease  ;  it  is 
not  communicable  from  man  to  man  under  natural  conditions.  The 
parasite  is  not  found  in  the  secretions,  but  it  appears  to  be  present  in 
the  herpetic  vesicles  of  malarial  patients,  because  the  contents  of  the 
vesicles  are  capable  of  transmitting  the  disease  by  inoculation.  The 
parasite  undoubtedly  exists  in  certain  swampy  districts,  endemic,  and  at 
certain  times  epidemic,  in  a  saprophytic  condition  in  either  the  earth, 
water,  or  air.  The  transmission  by  means  of  the  bite  of  insects  is 
theoretically  possible,  and  by  some  authorities  it  is  advanced,  but  it  has 
not  yet  been  satisfactorily  demonstrated.  The  incubative  period  in  the 
majority  of  cases  is  from  eight  to  fourteen  days  ;  but  cases  are  known 


SOUTHERN  FEVER  261 

where  the  disease  did  not  break  out  for  months,  and  others  where  it 
occurred  in  one  to  two  days,  or  occupied  only  a  few  hours.  These 
differences  are  probably  due  to  the  number  of  the  parasites  in  the 
system,  and  the  reaction  of  the  affected  individuals  to  the  poison,  or  to 
varieties  at  present  unknown  in  the  stages  of  the  parasite  outside  the 
body. 

SOUTHERN   OR  TEXAS   CATTLE  FEVER. 

(Ixodic  Anaemia  in  Jamaica,  Williams.       Australian   Tick    Fever.) 

This  disease  is  due  to  the  ravages  of  a  blood  parasite,  the 
'Pyrosoma  bigeminum, '  originally  discovered  by  Smith  in  the  red 
blood-corpuscles  of  cattle  affected  with  southern  fever.  Williams 
found  it  in  Jamaica,  and  Koch  has  also  recently  discovered  it  in 
Africa,  in  cattle  diseases  associated  with  the  presence  of  ticks  (Ixodes 
or  Boophilus  bovis). 

The  disease  affecting  cattle,  known  as  Texas  or  Southern  fever, 
commences  with  a  high  fever,  which,  unless  death  occurs  sooner, 
lasts  about  a  week,  and  is  associated  with  severe  anaemia  and 
haematuria;  a  chronic  condition  often  follows  when  the  symptoms 
are  not  so  exaggerated. 

Microscopical  Appearances  of  the  Parasite. — When  the  blood  of 
an  affected  animal  is  examined  in  the  fresh  state  at  ordinary  temperature, 
small  bodies  are  seen  inside  the  red  blood-corpuscles.  These  may  be  pale 
rounded  masses  with  amoeboid  movement  and  distinct  contour,  or  pear  or 
spindle-shaped  bodies,  distinctly  outlined,  with  a  granular  body  or  vacuole 
at  the  thick  end.  There  may  be  two  of  the  pear-shaped  bodies  with 
narrow  ends  opposed  in  a  single  corpuscle.  Very  rarely  three  or  four 
ovoid  forms  occur  in  the  same  corpuscle  (see  Photomicrograph,  Fig.  99). 
The  bodies  measure  from  J  to  2  /u,  in  diameter,  and  are  usually  situated  in 
the  disc  of  the  red  corpuscles. 

The  Motility  when  present  may  persist  for  an  hour.  In  the  blood 
circulation  the  infected  cells  rarely  exceed  1  to  2  per  cent.,  and  in  some 
cases  in  a  later  stage  the  parasite  may  be  found  free. 

If  an  animal  dies  or  is  killed  in  the  acute  febrile  stage  a  great 
number  of  infected  corpuscles  are  found  in  the  capillaries  of  the 
peripheral  circulation.  The  parasites  are  most  plentiful  in  the  vessels  of 
the  kidney,  next  in  the  liver  (see  Photomicrographs,  Figs.  99  and  100), 
spleen,  and  heart  substance. 

Staining  Reactions. — Cover-glass  specimens  heated  after  Ehrlich's 
method,  and  stained  with  alkaline  methylene  blue  one-half  to  two 
minutes,  washed  with  water,  and  placed  in  a  1  per  cent,  solution  of  acetic 


262  THE  PROTOZOA 

acid  for  a  few  seconds  and  then  washed  in  water  ;  examined  in  water,  or 
dried  and  mounted  in  xylol  balsam. 

Anatomical  Changes. — The  principal  post-mortem  lesions  observed  by 
the  author  during  an  extensive  outbreak  in  California  in  1888,  where  one 
(  rancher '  lost  700  out  of  a  herd  of  1200  in  from  three  to  four  weeks,  were 
as  follows.  The  blood  was  a  bright  brick-red  colour,  very  thin,  seeming 
to  coagulate  more  rapidly  than  normal  blood.  The  flesh  in  the  majority 
of  cases  was  of  a  bright  red  colour,  the  fat  between  the  muscles,  as  well 
as  that  of  the  alveolar  tissue,  of  a  deep  brownish  colour.  The  spleen  was 
enormously  enlarged,  of  a  dark  purple  colour,  the  capsule  sometimes  being 
studded  with  petechiae.  On  section  the  splenic  pulp  consisted  of  a  disin- 
tegrated mass,  having  lost  all  consistency,  pressure  causing  light  red  blood  to 
escape.  The  largest  measurement  obtained  was  24  in.  long  and  16  in.  in 
circumference.  The  stomach,  when  examined  in  situ,  shows  the  following 
appearances  : — The  reticulum  is  sometimes  the  seat  of  red  imbibitions.  The 
abomasum  is  always  the  seat  of  distinct  and  pathognomonic  changes.  The 
mucosa  often  presents  a  pink  or  dark-red  colour,  with  minute  ecchymoses 
studding  its  surface,  and  erosions  of  the  epithelium  are  also  frequently 
present.  The  duodenum  is  sometimes  of  a  deep  red  colour,  the  mucosa  deeply 
tinged  with  bile,  especially  close  to  the  pylorus.  The  jejunum  is  frequently 
reddened,  and  circumscribed  haemorrhagic  centres  are  often  seen.  The 
caecum  and  colon  are  generally  the  seat  of  more  or  less  ecchymoses.  The 
rectum  is  generally  of  a  red  colour,  and  when  diarrhoea  is  one  of  the 
symptoms,  the  mucosa  is  the  seat  of  extensive  haemorrhages.  The  liver  is 
the  most  affected  organ  of  any  in  the  body,  and  the  condition  of  it  may 
be  said  to  be  diagnostic  of  Southern  fever.  It  is  always  enlarged,  and 
in  most  cases  enormously  so,  records  showing  it  to  weigh  20  Ibs.,  and  in 
one  case  27  J  Ibs.  In  colour  it  is  light  brick-red,  inclining  to  a  dark  yellow, 
almost  resembling  powdered  cinnamon  ;  on  section  it  was  generally  fatty 
and  light  red  coloured  ;  blood  escaped  as  well  as  an  excess  of  bile.  Under 
the  capsule  yellow  streaks  could  be  seen,  some  as  large  as  straws,  due  to 
engorgements  of  the  bile-ducts.  Microscopical  examination  of  a  section 
of  such  reveals  a  condition  like  artificial  injection  of  the  gall  capillaries 
with  bile.  The  gall-bladder  was  in  all  cases  more  or  less  distended,  and 
in  some  as  large  as  a  urinary  bladder  ;  the  record  shows  it  to  have  weighed 
4  Ibs.  Its  walls  were  hypertrophied  and  full  of  gall,  sometimes  of  a  dark 
yellow,  and  again  of  a  dark  green  colour,  almost  black,  sometimes  slightly 
inspissated,  and  again  so  much  so  that  it  resembled  boiled  starch  in  consist- 
ency. The  kidneys  were  usually  of  a  dark  brown  colour,  from  the  intense 
congestion  ;  in  some  cases  they  were  enlarged,  in  others  not ;  there  was 
always  more  or  less  parenchymatous  degeneration  present,  and  diffuse 
capillary  engorgement.  The  cortex  was  softer  than  usual,  and  numerous 
petechiae  could  be  seen  throughout  its  substance.  In  some  cases  pus 
was  present  in  the  pelvis,  and  in  others  a  thin  yellow  exudate ;  the  fat 


SOUTHERN  FEVER  263 

of  the  kidney  had  the  same  peculiar  yellow  colour  already  described. 
The  bladder  was  usually  distended  with  bloody  urine,  and  in  such  cases 
the  mucosa  was  more  or  less  congested.  The  lungs  in  some  cases  were 
emphysematous,  in  others  a  broncho-pneumonia  was  present.  Again, 
some,  in  fact  all,  presented  a  more  or  less  hypostatic  pneumonia,  due  to 
weakness  of  the  heart  and  kidney  complications.  Pleurisy  was  also 
present  in  some  cases.  The  pericardium  was  studded  with  petechise  ;  the 
fat  around  the  heart  exhibited  the  same  peculiar  colour  already  mentioned. 
Serous  infiltrations  and  haemorrhages  were  sometimes  found  beneath  the 
skin  of  the  lower  jaw  and  neck. 

Mode  of  Infection. — A  ccording  to  Smith  and  Kilborne  the  natural 
infection  is  caused  by  the  blood-sucking  tick  (Ixodes  or  Boophilus  bovis) 
which  lives  on  the  skin  of  the  cattle,  in  an  enzootic  territory  becoming 
charged  with  the  parasite. 

The  mature  female  tick  on  the  animal's  body  has  an  oblong,  oval- 
shaped  body,  and  somewhat  resembles  a  castor-oil  bean ;  it  presents  a 
dull  leaden  colour,  is  rarely  more  than  J  an  inch  long,  and  -f$  of  an  inch 
in  breadth,  possessing  four  pairs  of  legs,  situated  on  the  antero-lateral 
portion  of  the  body.  It  attaches  itself  to  its  host  by  what  is  known  as 
the  rostrum,  in  the  centre  of  which  is  a  barbed  dart  furnished  on  either 
side  with  several  rows  of  teeth  arranged  obliquely,  which  enable  the 
tick  to  adhere  more  securely  to  the  skin.  The  male  tick  is  usually 
found  attached  to  the  skin  immediately  underneath  the  anterior  portion 
of  the  female,  the  body  being  of  a  dark  brown  colour  and  triangular  in 
shape.  When  fully  grown  it  is  very  much  smaller  than  the  fully 
developed  female,  and  never  being  in  an  engorged  condition,  is  much 
more  active  and  stronger  in  comparison  with  the  female.  As  the  female 
tick  engorges  herself  she  is  fecundated  by  the  male,  and  at  maturity 
withdraws  the  barbed  mouth  organs  or  rostrum,  falls  to  the  ground,  lays 
a  great  number  of  eggs,  shrivels  up  gradually  and  perishes.  The  para- 
site is  carried  over  in  the  eggs,  and  in  from  two  to  six  weeks  the  young 
ticks  are  hatched,  creep  upon  the  cattle,  and  infect  them  with  the 
disease.  By  this  means  fresh  cattle  arriving  in  infected  districts 
become  affected,  while  the  native  cattle  exhibit  no  diseased  symptoms. 
It  is  possible  that  they  were  infected  as  calves,  and  since  then  continued 
to  harbour  the  blood  parasites ;  in  fact,  inoculation  of  the  blood  of  such 
animals  produces  the  infection  in  other  cattle  in  regions  where  the 
disease  does  not  exist.  Cattle  travelling  from  infected  regions  can 
infect  healthy  pastures,  not  by  direct  infection,  but  in  a  round-about 
manner.  The  ticks  that  the  cattle  bring  with  them  fall  from  the 
animals  and  deposit  their  eggs  on  the  ground,  and  the  young  ticks 
infect  the  previously  healthy  cattle,  the  disease  breaking  out  in  forty- 
five  to  sixty  days  after  the  appearance  of  the  infected  cattle,  because 
so  much  time  must  elapse  before  the  young  brood  of  ticks  is  capable  of 


264  THE  PROTOZOA 

producing  the  disease.  The  infection  can  also  be  conveyed  to  healthy 
districts  by  the  artificial  dissemination  of  mature  forms  of  the  ticks, 
which  does  not  take  place  when  the  ticks  are  removed  from  the  infected 
cattle  before  they  are  driven  on  new  tracts  of  land.  The  Pyrosoma 
bigeminum,  in  certain  respects,  is  somewhat  similar  to  the  malaria 
organism,  but  is  distinguished  by  dividing  into  two,  and  producing  no 
pigment.  Although  many  particulars  in  their  development  are  not  suffi- 
ciently cleared  up,  their  place  with  the  amoeba  is  certain. 

Immunity. — Koch  recently  stated  in  his  address  to  the  Colonial 
Society,  Berlin,  that  he  was  able  to  infect  healthy  cattle  with  ticks 
taken  from  diseased  ones,  and  had  succeeded  in  conferring  immunity 
against  Texas  fever  as  follows  : — Healthy  animals  were  inoculated 
with  the  blood  of  the  animals  infected  by  young  ticks,  and  Texas 
fever  produced,  and  the  process  continued  for  several  generations, 
until  the  disease  was  produced  in  a  number  of  animals  which  re- 
covered. These  recovered  animals  were  infected  a  second  time  with 
the  blood  of  sick  animals,  being  immune  to  the  mild  form  of  the 
disease  produced  under  the  conditions  of  the  experiment.  The 
immune  animals  were  next  taken  to  the  coast  and  turned  out  with 
an  affected  herd,  but  did  not  become  affected.  They  were  finally 
inoculated  with  the  blood  of  an  animal  affected  with  a  severe  form 
of  the  disease,  when  only  one  of  the  immunized  animals  became 
affected. 


BABESIA   BOVIS   (STARCOVICI). 

This  parasite  was  first  found  by  Babes  in  the  red  blood-corpuscles 
of  the  blood  of  cattle  affected  with  hsemoglobinuria  in  Roumania. 

Microscopical  Appearances. — It  is  found  throughout  the  vascular 
system,  either  as  round  or  lanceolate-shaped  bodies,  mostly  lying  in  pairs 
or  undergoing  a  process  of  division,  and  staining  like  bacteria.  In  the 
fresh  state  they  are  non-motile,  and  measure  about  1  />t,  and  in  stained 
specimens  about  0'6  /x  in  diameter.  In  the  commencement  of  the 
attack  large  pear-shaped,  irregular,  staining  bodies  were  also  observed. 

The  symptoms  of  the  disease  in  the  affected  cattle  are  similar  to 
those  of  Southern  fever. 

Haematuria  is  frequent  during  the  life  of  the  animal,  the  attack 
lasting  about  five  days. 

The  chronic  stage  sometimes  occurring  in  Texas  fever  was  not 
observed.  According  to  Babes,  the  ticks  play  an  important  part  in  the 
production  of  the  Roumanian  disease,  the  blood  parasites  increasing  in 
the  bodies  of  the  ticks. 


FIG.  99.— Pyrosoma  Bigeminum  in  bloodvessel.    Section  of  liver 
of  affected  ox.    Methylene  blue.     X  600. 


Fio.  100.— Pyrosoma  Bigeminum  in  bloodvessel.    Section  of 
liver  of  affected  ox.    Methylene  blue.     X  1000. 


[T.  BmuUll,  F.R.C.V.S.,  Photo.,  Edinburgh,  1898. 


FIG.  101.— Klossia.    Section  of  liver  of  snail  (AquoHmax.) 

xiooo. 


T.  Bowhill,  F.R.C.Y.S.,  Photo.,  Edinburgh,  1898. 


BOVINE  MALARIA  265 


BABESIA   OVIS   (STARCOVICI). 

This  parasite  is  the  cause  of  ictero-haemoglobinuria  of  Roumanian 
sheep,  and  was  also  found  by  Bonome  in  an  outbreak  in  Italy.  The 
transmission  of  affected  blood  to  healthy  sheep  in  large  quantities 
yielded,  according  to  Babes  and  Bonome,  no  genuine  reproduction  of 
the  disease. 

BOVINE  MALARIA. 

Celli  and  Santori  describe  a  disease  amongst  cattle  in  the  Roman 
Campagna  which  they  call  cattle-malaria.  The  disease  is  characterised 
by  acute  anaemia,  enlargement  of  the  spleen,  emaciation,  and  fever. 
Native  cattle  do  not  appear  to  be  affected  with  the  disease,  but  Swiss, 
Lombardy,  and  Dutch  cows  are  affected,  and  sometimes  death  results. 
In  the  blood  of  the  affected  animals  parasites  were  found  inside  the  red 
blood  corpuscles,  which  sometimes  exhibited  Brownian  and  at  other  times 
amoeboid  movement.  The  endoglobular  parasites  were  sometimes  pear- 
shaped  and  united  in  pairs,  and  identical  with  Smith's  Pyrosoma  bigeminum 
of  Texas  fever. 

Celli  and  Santori  consider  this  malady  is  probably  identical  with 
diseases  described  by  other  observers,  while,  on  the  other  hand,  they  find 
many  resemblances  between  this  disease  and  human  malaria.  Quinine 
administered  to  the  affected  cattle  produced  favourable  results. 

KLOSSIA   SOROR  (A.  SCHNEIDER). 

This  parasite  is  frequently  found  in  the  kidneys  of  land  and  water 
snails  (Helix,  Succinea,  Neritina,  etc.),  and  probably  also  in  other 
species. 

Microscopical  Appearances. — Cysts  containing  a  large  number  of 
permanent  spores.  Each  of  these  mother  spores  divides  into  four  to  six 
sickle-shaped  embryo  (or  daughter  spores).  The  sickle-shaped  spores 
are  1  to  7  ft  in  size,  and  exhibit  a  serpentine  motility  for  a  short  time. 
The  daughter  spores  often  increase  in  the  epithelial  cells  of  the  kidney, 
causing  hypertrophy  of  the  same.  Sickle  forms  have  also  been  observed 
in  the  secretion  of  the  kidneys.  (For  Photomicrograph  of  Klossia  in  the 
liver  of  a  snail,  see  Fig.  1 06.) 

PHOTOMICROGRAPHY. 

The  four  plates  of  photomicrographs  produced  at  the  end  of  this 
work  were  made  with  the  large  Zeiss  Photomicrographic  Apparatus  (see 


268  PHOTOMICROGRAPHY 

Fig.   102)  with  a  Schuckert  &  Co.  electric  lamp,  enabling  the  work  to 
be  done  at  any  time. 

Between  the  electric  lamp  and  the  microscope  the  following  articles 
are  placed  in  a  direct  line  on  a  sliding  bar : — 

1 .  A  Collecting  Lens,  diameter  4^  inches,  consisting  of  an  anterior  and 
posterior  lens.     The  concave  surfaces  of  these  lenses  are  turned  towards 
the  light,  and  adjusted  so  that  the  distance  between  the  luminous  centre 
and  the  edge  of  the  lens  mount  is  about  4f  inches,  rendering  the  rays 
nearly  parallel,  without  introducing  excessive  aberrations.     The  anterior 
lens  is  placed  close  to  the  lamp,  and  the  posterior  lens,  which  again 
collects  the  parallel  rays,  on  a  point  situated  about  16  inches  from  the 
anterior  lens  (the  collecting  lenses  can  be  rotated). 

2.  Between  the  anterior  and  posterior  lenses    an   absorbing   cell   is 
placed  for  the  absorption  of  the   heat  rays  generated  by  the  electric 
lamp.     The  walls  of  the  cell  consist  of  plate-glass  discs  about  4|  inches 
in  diameter,  while  the  distance  between  the  glass  discs  is  about  2 \  inches  ; 
an  inlet  tap  is  fitted  on  the  bottom  and  an  outlet  tap  on  the  top  of  the 
body  of  the  cell.     (This  cell  can  also  be  used  with  sunlight.)     The  cell 
is  filled  with  water,  previously  boiled,  from  the  lower  tap,  because  if 
filled  from  above  the  almost  inevitable  result  is  cracking  of  the  glass 
discs. 

3.  An   Iris   diaphragm   is    placed    between    the    microscope    and    the 
posterior  collecting  lens,   with   a  total  aperture  of  2|  inches,  fitted  with 
spring  clips  for  holding  objects,  coloured  screens,  and  ground  glass  for 
indirect  illumination. 

The  iris  diaphragm  is  particularly  adapted  for  centering  the  entire 
apparatus,  for  shutting  off  false  light,  and  when  closed  the  illuminating 
rays  are  focused  in  the  small  opening  of  the  closed  diaphragm  (about 
J  an  inch)  by  means  of  the  posterior  collecting  lens. 

4.  The  microscope  used  with  the  large  Zeiss  apparatus  is  their  special 
stand    for  photomicrography    (see    Fig.    103),    which   is    placed    on   an 
adjustable  sole  plate  with  levelling  screws,  and  instead  of  the  ordinary 
Abbe  condenser,  a  special  centering  achromatic  condenser  is  used  (see 
Fig.  104),  with  an  aperture  of  TO,  and  is  focused  with   respect  to  the 
plane  of  the  object  by  means  of  the  rack  and  pinion  movement  of  the 
illuminating   apparatus.      The    aperture    of  the   illuminating   pencil  is 
regulated  by  an  iris  diaphragm  placed  between  the  lenses  of  the  con- 
denser.     The  best   lenses   for  photomicrographic  work,   especially  for 
bacteria  and  other  high-power  photography,  are  the  apochromatic  lenses 
of  Zeiss.       The   photomicrographs   in   this    work,    reproduced    by   the 
colotype    process,  were    made    with    the    apochromatic    homogeneous 
immersion  lens  2  mm.,  numerical  aperture  1'40,  while  those  reproduced 
by  half-tone  process  were  made  with  a  1-5  mm.  lens  with  a  numerical 
aperture  of  1*30,  and  illuminated  by  means  of  a  special  heliostat  of  my 


PHOTOMICROGRAPHY  269 

own  construction.  Projection  eye-pieces,  Nos.  2  and  4  (see  Fig.  105) 
were  used  in  conjunction  with  these  lenses. 

The  microscope,  electric  lamp,  and  other  fittings  connected  therewith, 
just  described,  are  placed  on  a  slide  on  a  table  as  shown  in  Fig.  102, 
while  the  camera  is  placed  on  a  separate  stand  (see  Fig.  102). 

For  sunlight  a  light  filter  is  used,  while  with  the  electric  light 
spectroscopic-tested  glass  plates  are  used — for  red  stained  specimens, 
green ;  for  blue  stained  specimens,  yellow ;  for  violet,  yellow,  and  green, 
and  for  unstained  specimens,  blue. 

For  focusing  the  image  on  the  ground  glass  of  the  camera  a  special 
improved  aplanatic  focusing  lens  is  used  with  an  exceptionally  large 
field  and  a  magnification  of  6 — a  higher  power  is  a  disadvantage.  The 
fine  adjustment  during  focusing  is  manipulated  by  means  of  a  Hook's 
key  and  rod  connection. 

Magnification. — This  depends  upon  the  distance  of  the  specimen  from 
the  ground  glass  slide,  and  can  be  accurately  measured  as  follows  : — Take 
a  stage  micromillimeter  (which  is  one  millimeter  divided  into  hundredths)  ; 
place  it  under  the  lens  and  project  the  picture  on  the  ground  glass  slide  ; 
adjust  the  focus,  and  measure  the  size  with  an  ordinary  glass  100  milli- 
meter measure.  When  1  millimeter  of  the  projected  stage  micrometer 
is  equal  to  10  millimeters  on  the  glass  measure,  then  the  magnification  = 
1000  diameter.  Other  magnifications  are  calculated  in  a  similar  manner. 


APPENDIX 


KISCHENSKY'S    QUICK    METHOD    OF    STAINING    BACTERIA 
IN  COVER-GLASS  AND  SLIDE  PREPARATIONS. 

1.  A  drop  of  a  weak  solution  of  carbol  fuchsin  (10  drops  to  10  c.c.  of 
water)  is  placed  on  a  cover-glass  and  mixed  with  a  minimum  quantity  of 
the  pure  culture  under  investigation,  and  spread  out  in  a  thin  layer. 

2.  The  preparation  is  now  heated  gently  over  the  flame. 

3.  In  examining  blood,  pus,  etc.,  the  above  stain  is  mixed  with  an 
alcoholic  solution  of  methylene  blue. 

The  results  are :  the  quickness  of  the  method,  beautiful  staining,  by 
which  means  the  flagella  can  also  be  demonstrated,  while  the  field 
remains  unstained. 


SEMENOWICZ  AND  MARZINOWSKY'S  SPECIAL  METHOD  OF 
STAINING  BACTERIA  IN  COVER-GLASS  SPECIMENS  AND 
SECTIONS. 

1.  The  cover-glass  specimens  and  sections  are  stained  two  (three  to 
four)  minutes  in  a  solution  of  carbol  fuchsin  (one  part  of  a  concentrated 
solution  and  two  parts  of  water). 

2.  The  specimens  are  now  washed  with  water. 

3.  Stain  three  to  four  (four  to  five)  minutes  with  Loffler's  methylene 
blue. 

4.  Wash  in  alcohol. 

5.  Clarify  in  oil  and  xylol. 

Results. — Loffler's  blue  displaces  the  carbol  fuchsin  in  the  bacteria 
and  appears  to  work  on  the  principle  of  a  mordant.  The  nuclei  and 
the  bacteria  are  stained  blue,  the  interstitial  tissue  and  the  protoplasm 
of  the  cells  red,  while  degenerated  bacteria  also  appear  red. 

GASTROMYCOSIS   OVIS   (NIELSEN). 

This  organism  was  isolated  in  a  very  rapidly  fatal  endemic  disease 
amongst  sheep  in  Iceland  and  Norway,  characterised  by  a  haemorrhagic 


272  APPENDIX 

inflammation  of  the  abomasum  and  the  other  compartments  of  the 
stomach,  portions  of  the  intestines,  as  well  as  the  whole  body.  The 
bacilli  were  found  in  the  walls  of  the  stomach  and  frequently  in  the 
internal  organs  three  hours  after  death.  This  disease,  from  the 
description  given  by  Nielsen,  is  probably  identical  with  a  disease 
affecting  sheep  in  the  country  known  as  '  Braxy.' 

Microscopical  Appearances. — Bacilli  from  1  to  2  by  6  p,  often 
occurring  in  pairs,  seldom  arranged  in  chains,  frequently  containing 
refractile  bodies. 

Staining  Reactions. — Stains  by  the  Gram  method. 

The  bacillus  has  not  been  cultivated  on  artificial  media.  Experiments 
conducted  with  pieces  of  tissue  containing  the  bacilli,  introduced  into 
rabbits  and  lambs,  as  well  as  feeding  experiments,  yielded  negative 
results. 

SPECIAL   BACILLUS    ISOLATED   FROM   MILK   IN 
PIORKOWSKI'S   LABORATORY,  BY   CAMPBELL   M'CLURE 

(Deutsche  Med.  Wochemchrift,  1898,  No.  26). 

Microscopical  Appearances. — It  occurs  as  short  rods  a  little  shorter 
and  thicker  than  the  diphtheria  bacillus.  Club-shapes  are  often  observed, 
especially  in  old  cultures. 

Motility. — Non-motile. 
Spore  Formation  absent. 

Staining  Reactions. — Stains  with  any  of  the  ordinary  dyes,  but  not 
by  the  Gram  method. 

Biological  Characters. — On  Gelatine  Plates. — In  two  to  three  days 
a  luxuriant  growth  develops  on  the  surface  of  the  medium  of  small  white, 
slightly  transparent,  granular -points,  with  well-defined  borders,  becoming 
later  (in  forty-eight  hours)  yellowish-brown  in  colour,  dark  in  the  middle, 
and  clear  at  the  periphery. 

In  Gelatine  Slab  Cultures  opaque  white  points  develop  along  the 
inoculation  track,  tending  to  become  confluent  in  the  centre. 

On  Agar  Plates  at  37°  C.,  light  brown  granular  colonies  develop. 

On  Glycerine  Agar,  a  similar  growth. 

Bouillon  at  37°  C.  becomes  cloudy,  a  flaky  sediment  forming  on  the 
bottom  and  sides  of  the  tube. 

On  Potatoes,  at  37°  C.  a  copious,  greyish-white,  moist  coating,  with 
dentated  edges,  which  become  yellow  in  older  cultures. 

On  Grape  Sugar  Agar  Media  the  growth  exhibits  no  special 
characteristics. 

Milk  inoculated  and  kept  at  37°  C.  for  forty-eight  hours  has  an  acid 


APPENDIX  273 

reaction,  is  coagulated,  and  emits  an  odour  of  acetic  acid  (this  feature  is 
not  constant). 

Pathogenesis. — A  mouse  inoculated  subcutaneously  died  in  fourteen 
days.  Investigation  of  the  heart's  blood,  spleen,  liver,  and  lungs 
yielded  negative  results. 

BACELLFS  METHOD  OF  TREATING  TETANUS  IN  MAN. 

Bacelli  injects  subcutaneously  3  to  4  centigrammes  of  a  2  to  3  per 
cent,  solution  of  carbolic  acid  until  about  35  centigrammes  have  been 
injected  in  the  twenty-four  hours. 

The  carbolic  solution  acts  as  curative  serum  on  the  tetanus  poison 
circulating  in  the  blood,  besides  on  the  nerve  elements  already  affected 
by  the  poison.  The  fact  that  so  large  a  quantity  of  carbolic  solution  can 
be  taken  into  the  system  without  appearances  of  intoxication,  the  author 
declares  to  be  due  to  a  special  resistance  of  the  tetanic  poison  against 
the  remedy — (Munich  Meet.  Woch.,  Dtsck.  Med.  Zg.) 

List  of  some  of  the  Principal  Works  consulted  in  the 
Compilation  of  the  Manual. 

BOOKS. 

C.  FLUGGE — '  Die  Micro-organism.' 

C.  GUNTHER. — '  Einfuhrung  in  das  Studium  der  Bacteriologie.' 

SCHURMAYER. — ( Bacteriologische  Technik.' 

FRAENKEL. — '  Bacteriologie.' 

KALDEN'S  '  Technik.' 

LEHMANN  and  NEUMANN. — ( Bacteriologie.' 

BOHM  and  OPPEL. — <  Mikroskopischen  Technik.' 

LEVY  and  KLEMPERER. — (  Bacteriologie.' 

KITT. — '  Atlas  der  Thierkrankheiten.' 

FRIEDLANDER  and  EBERTH'S  '  Mikroskopische  Technik.' 

ROBERT  BEHLA. — ( Die  Amoben.' 

'  Atlas  der  Klinischen  Mikroskopie  des  Blutes.' 

MOSSELMAN  and  LIENAUX. — '  Veterinary  Microbiology.' 

CORNEL  and  BABES. — c  Les  Bacteries.' 

WOODHEAD. — '  Bacteria  and  their  Products.' 

ABBOT. — '  Principles  of  Bacteriology/ 

KANTHACK  and  DRYSDALE. — (  Practical  Bacteriology.' 

STERNBERG. — '  Manual  of  Bacteriology.' 

KLEIN. — (  Micro-organisms  and  Disease.' 

SIMON. — (  Clinical  Diagnosis.' 

LAFAR. — '  Technical  Mycology.' 

S 


274  APPENDIX 

SYKES. — 'Principles  and  Practice  of  Brewing/ 

NEUMAN. — '  Parasites  and  Parasitic  Diseases  of  Domestic  Animals/ 

BILLINGS. — '  Investigations  in  Nebraska/ 

PERIODICALS. 

'  Hygienische  Rundschau/ 
'  Centralblatt  fiir  Bacteriologie/ 

'  Archiv  fiir  Wissenschaftliche  und  Praktische  Thierheilkunden/ 
'  Berliner  Thierartz  Wochenschrift/ 
'Recueil  de  Medecine  Veterinaire/ 
'  The  Lancet/ 
'  British  Medical  Journal/ 
'Science  Progress/ 
'The  Veterinary  Record/ 
'The  Veterinarian/ 
'The  Veterinary  Journal/ 

'  Journal  of  Comparative  Pathology  and  Therapeutics  ' — Reports  of 
the  U.S.  Bureau  of  Animal  Industry. 


Plate  I 


* 

X 


V 


I. 

Bacillus  Anthracis    with    capsules    in 

Mouse's  blood  stained  by  Johne's  Method 

X  1.000. 


III. 

Bacillus  Anthracis  section  of  Mouse's 
lung.  Methylene-blue  X  350. 


II. 


Bacillus   Anthracis    and  Spores    Agar 
Culture  Fuchsin  X  1.000. 


*«    .,  '^.v^'^fc^v^-1  i'i-% 

ssaSRlk 


Bacillus  of  Symptomatic  Anthrax  with 
flagella.  Grape  -  sugar  -  agar  culture 
stained  with  Orcein  Solution  X  1. 000. 


v. 

Bacillus  of  Malignant  Oedema  with 
fiagella.  Grape  -  sugar  -  agar  culture 
stained  with  Orcein  Solution  X  1.000. 


/^ 


VI. 

Bacillus  of  Tetanus  with  flagella.  Grape- 

sugar-agar  culture  stained  with  Orcein 

Solution  X  1.000. 


fiowhill,  photo.    Berlin  1898. 


Collotype  by  C.  G.  Roder,  Leipzig. 


Plate 


VII. 

Bacillus  of  Typhoid  Fever  with  flagella. 
Agar  culture  stained  with  Orcein  So- 
lution X  1.000. 


.          . 

Mfc-     I 


VIII. 

Bacillus  of  Typhoid  Fever  with  flagella. 
Agar  culture  stained  with  Orcein  So- 
lution X  1.500. 


Finkler  and  Prior's  Spirillum  with  fla- 
gella.  Agar  culture  stained  wTith  Orcein 
Solution  X  1.000. 


%&m 


mm 


XI 

Bacillus  Coli  communis  with  flagella. 
Agar  culture  stained  with  Orcein  So- 
lution X  1.000. 


Cholera    Spirilla    with  flagella.     Agar 

culture  stained    with    Orcein  Solution 

X  1.000. 


XII. 

Proteus  Vulgaris  with  flagella.     Agar 

culture  stained   with    Orcein  Solution 

X  1.000. 


Bowhill,  photo.    Berlin  1898. 


Collotype  by  C.  G.  Roder,  Leipzig. 


Plate  III 


. 
-  ••-••*•,-•••.•    •  •  • 

•     .    •    -    .':'>  -\i*    - 


*V   iv  ^  * 

v*  • 

•      \  *%      *   • 

.V  ./    g    % 


XIII.  ' 

Bacillus  of  Swine  Fever  with  flagella. 
A  gar'  culture  stained  with  Orcein  So- 
lution X  1.000.     - 


XIV. 

Bacillus  of  Schweine-Seuche  "German". 
Agar  culture' stained  with  Orcein  So- 
lution X  1.000. 


I  l 


'^jff 


XV. 

Bacillus  of  Mouse  septicaemia  in  Mouse's 
blood.  "Cladius  stain".  X  1.000. 


XVI. 

Bacillus  'of  Glanders.     Glycerine  Agar 
culture  Fuchsin  X  1.000. 


! 


XVII. 

Bacillus    of  Leprosy    section    of  skin- 
nodule    "Cladius  stain"  X  1.000. 


XVIII. 

*)  Bacillus    of   Tuberculosis    X    1.000. 

Cover  glass  preparation  from  a  Guinea 

pig  inoculated  with  a  culture  from  a 

Guinea  pig  inoculated  with  butter. 


*)  For  the  specimen  from  which  this  photograph  was  obtained  I  am  indebted 
to  Drs.  Morgenroth  and  Hormann,  Hygienic  Institute,  Berlin. 


Bowhill,  photo.    Berlin  1898. 


Collotype  by  C.  G.  Roder,  Leipzig. 


Plate  IV. 


XIX.  XX. 

Bacillus    of   Diphtheria    Blood- Serum.  Bacillus  of  Bubonic  Plague  in  human 

Agar  culture   uCladius  stain"  X  1.000.  blood  X  1.000. 


XXI. 

Fraenkels    Diplococcus  of  pneumonia. 
Agar  culture  Fuchsin  X  1.000. 


XXIII. 

Schiitz's  Streptococcus  of  Strangles  in 
pus  from  abscess  Cladius  stain  X  1.000. 


J 
XXII. 

Staphylococcus  pyogenes  aureus.    Agar 
culture  "Cladius  stain"  X  1.000. 


s 

^ 


>>"> 

XXIV. 

Gonococci,  in  urethral  discharge  stained 
by  Knacks  Method  X  1.000. 


Bowhill,  photo.    Berlin  1898. 


Collotype  by  C.  G.  Roder,  Leipzig. 


INDEX 


ABBOT'S   method    of    describing  an   or- 
ganism, 85 

Abscesses,  cause  of,  88 
Achorion  Schonleinii,  235 
Acid-resisting  bacteria,  137 

tubercle-like  bacilli  in  butter,  142 

method  of  demonstrating,  143 
Acne  contagiosa  of  horses,  101 

man,  102 
Actinomyces  bovis,  102 

hominis,  106 

musculorum  suis,  106 
Agar-agar  media,  ordinary,  53 

grape  sugar,  54 

glycerine,  54 

Wurtz's  lactose  litmus,  54 

blood,  54 

gelatine,  55 

Agents,  differentiating,  45 
Air,  bacteria  in,  206 

examination  of,  ordinary  method,  80 
Hesse's  method,  80 
Petri's  method,  80 
Amoeba  coli,  254 
Anaerobic  culture  methods,  64 

Buchner's,  66 

Fraenkel's,  65 

Hesse's,  65 

Kasparee's,  67 

Liborious's,  65 
Anilin  dyes,  14,  40,  41,  4-> 

oil,  42,  45 

water,  42 

Animal  parasites,  254 
Anthrax,  bacillus  of,  109 

antidotal  effect  of  B.  pyocyanus  in,  97 

bacteriological  diagnosis  of,  112 

differential  diagnosis,  113 
table,  122 

in  man,  111 

involution  forms,  1 10 

NuttalPs  insect  experiments  with,  112 

symptomatic!,  114 
Antitetanic  serum,  119 
Ascococcus,  characters  of,  4 

Billrothii,  224 
Asiatic  cholera,  bacillus  of,  153 

bacteriological  diagnosis  of,  156 


Asiatic  cholera,  bacillus  of,  examination 
of  water  for,  157 

specific  reactions,  155 
Aspergilli,  characters  of,  238 

in  pseudo-tuberculosis,  151 
Aspergillus  albus,  240 

clavatus,  240 

flavescens,  or  flavus,  240 

fumigatus,  240 

glaucus,  240 

nidulans,  239 

niger,  240 

ochraceus,  240 

oryzae,  240 

repens,  240 

subfuscus,  240 
Australian  tick  fever,  261 
Avian  tuberculosis,  148 

B 

BABESIA  bovis,  264 

ovis,  265 

Bacilli,    general    characters    and    mor- 
phology, 4 
Bacillus  acidi  lactici,  216 

acid-resisting  tubercle-like,  142 

aceticus  (Hansen's),  220 

of  acne  contagiosa  of  horses,  101 

amylobacter,  221 

anaerobius,  Flugge,  II.,  III.,  IV.,  214, 
215 

anthracis,  109 

symptomatici,  114 

aquatilis,  209 

arborescens,  208 

argento  phosphorescens,  225 

auranticus,  206 

of  blue  milk,  215 

botulinis,  123 

bovisepticus,  179 

of  broncho-pneumonia  bovis,  200 

brunneus,  207 

of  bubonic  plague,  196 

buccalis  maximus,  205 

butyricus,  221 

of  canary  septicaemia,  184 

capsulatus,  227 

cholerae  Asiatics,  153 
columbarum,  181 

of  cholera  in  chickens,  182 

of  cholera  in  ducks,  181 


276 


INDEX 


Bacillus  coli  communis,  174 
constrictus,  206 
coprogenes  foetidus,  194 
cyanogenus,  215 
diphtheria,  162-167 

colurabarum,  167,  168 

vitulorura,  168 
dysenteries  vitulorum,  180 
enteritidis,  125 
equi  intestinalis,  176 
erysipelatis  suis,  193-195 
felis  septicus,  187 
figurans,  128 
fluorescens  aureus,  207 

liquefaciens,  208 

non-liquefaciens  immobilis,  207 
fuscus,  207 

of  gastromycosis  ovis,  271 
glanders,  129-134 
glischrogenus,  206 
of  grouse  disease,  184 
gumosus,  219 
helvolus,  208 
of  hog  cholera,  188 
ianthinus,  208 
of  influenza,  176-179 
pseudo-influenza,  179 
lacticus,  216 
lactis  acidi,  216 
lactis  'Flugge,'  217 

inocuus,  217 

albus,  218 

Bleischii,  218 

erythrogenes,  218 

pituitosi,  218 
leprse,  152 

limbatus  acidi  lactici,  219 
liquefaciens,  209 

bovis,  201 

liquidus,  209 

malignant  oedema,  113,  114 
mallei,  129 
megaterium,  228 
mesentericus,  210 

fuscus,  210 

ruber,  210 

vulgatus,  210 
morbificans  bovis,  126 
of  mouse  septicaemia,  195 
murisepticus,  209 
mycoides,  209 
Neapolitanus,  174 
oedematis  maligni,  113,  114 
orchiticus,  134,  135 
Pasteurianus,  220,  221 
phasiani  septicus,  183 
phosphorescens,  225,  2-26 

indicus,  226 

indigenus,  226 

of  pneumonia,  '  Friedlander,'  92,  93 
prodigiosus,  208,  209 
proteus  fluorescens,  128 

mirabilis,  127,  129 

vulgaris,  127-129 


Bacillus  of  proteus  Zenkeri,  127,  129 
pseudo-tuberculosis,  151 

typhoid,  173,  207 
pyocyanus,  96 
radicicola,  231 
of  rouget,  193-195 
rubefaciens,  207 
rubidus,  208 

septicaemias  haemorrhagicse,  179-201 
smegmatis,  137,  152,  153 
spinosus,  210 
subflavus,  207 
subtilis,  210 
suisepticus,  208 
swine  erysipelas,  193 

fever,  188 
of  swine  plague,  188 

symptomatic  anthrax,  114-116 

syphilis,  137 

pneumonia  of  turkey,  185,  186 
tetani,  116-121 
Thermophilus,  226 
Trommelschlager,  227 
tuberculosis,  136-148 

avian,  148,  149 
tubergenus  III.  and  V.,  231 
typhi  abdominalis,  169-174 

murium,  187,  188 

ureae,  205,  206 
violaceus,  208 
viscosus,  208 
viscosus  cerevisiae,  222,  223 

lactis,  219,  220 

sacchari,  223 

vini,  223 
zeae,  219 

Bacteria,  acid-resisting,  137 
aerobic,  2 
aero-anaerobic,  3 
anaerobic,  2 
in  air,  206-213 
causing  acetic  acid  fermentation,  220 

butyric  acid  fermentation,  221 

specific  changes  in  beer,  urine,  and 
sugar,  222-224 

ropiness  in  milk,  219,  220 
chromogenic,  2 
classification  of,  3,  4 
culture  methods,  58 
denitrifying,  2 
dimensions  of,  5 
drumstick,  227 
effect  of  light  on,  3 

temperature  on,  3 
facultative,  3 
filter,  73 

in  inflammation  and  suppuration,  87 
involution  forms,  5 
in  leguminous  nodules,  230 
in  meat  poisoning,  122 
in  milk,  213-219 
morphology  of,  3,  4 
motile  organs  or  flagella  of,  5 
in  mouth,  205 


INDEX 


277 


Bacteria,  multiplication  of,  5 

in  nephritis,  206 

nitrifying,  characters  of,  2 

nitrifying,  232 

photogenic,  definition  of,  2 

pleomorphism  of,  5 

points  to  be  observed  in  describing, 
85,  86 

pyogenic,  definition  of,  2 

saprogenic,  definition  of,  2 

in  soil,  206-213 

special  reactions  during  growth,  68 

stained  by  Gram's  method,  19 

staining  capsules  of,  25 

thermophilic  characters,  3,  226 

thiogenic  definition  of,  2 

in  urine,  205,  206 

in  water,  206-213 

zymogenic  definition  of,  2 
Bacteriological  technique,  13-86 

general  methods  of,  15 

reagents  for  daily  use,  14 

stains  for  daily  use,  14 

working  bench  accessories,  etc.,  13,  14 
Bacterium  acidi  lactici,  219 

Peter's,  220 

aeruginosum,  96 

coli  commune,  174-176 

phosphorescens,  225 

Zopfii,  228 

Barbone  dei  bufali,  179 
Beggiatoa,  230 
Billing's  swine  plague,  188 

immunity  in  do.,  189 

corn-stalk  disease,  200 
Black  quarter,  114 
Blastomycetes,  1,  243 
Blenorrhoea  neonatorum,  95 
Blood,  examination  of,  28,  29 

serum  media,  fluid  and  solid,  55 
quick  method  of  preparing,  56 
Loffler's  do.,  56 

Booker's  roll  culture  method,  61 
Boophilus  bovis,  261,  263 
Botriococcus  ascoformans,  100 
Botriomycosis,  100,  101 
Botrytis  tonsurans,  237 
Botulismus,  122-125 

bacteriological  diagnosis  of,  125 
Bouillon,  preparation  of,  47 

glycerine,  48 

grape  sugar,  48 

milk  sugar,  48 
Bovine  farcy,  108 

tuberculosis,  140 

in  meat  and  milk,  141 
Bowhill's  stain  for  flagella  and  bacteria, 

43 

Braxy,  272 
Breustseuche,  99 
Broncho-pneumonia  bovis,  200 
Bubonic  plague,  bacillus  of,  196-200 

vitality  of,  197 

Nuttall's  experiments  with,  198 


Bubonic  plague,  bacteriological  diagnosis 

immunity  in,  199 
Haifkine's  serum,  199,  200 
Buchner  method  for  anaerobic  cultures, 

66 
Butter,  acid-resisting  bacteria  in,  141- 

144 

bacillus  tuberculosis  in,  141,  144 
Roth's  method  of  examining  for  bac- 
teria, 25 


CANARY  bird,  septicaemia  of,  184,  185 
Canine  distemper,  204,  205 

tuberculosis,  145 
Capsule  bacilli,  4 
cocci,  4 

method  of  staining,  25 
Catarrh,  92 
Cattle  malaria,  265 
Cerebral  tetanus,  121 
Charbon,  109 

symptomatique,  114 
Chicken  cholera,  182,  183 

disease,  proteus  fluorescens  in,  128 
Choleras  Asiaticae,  153-158 
of  chickens,  182,  183 

ducks,  181,  182 
infantum,  129 
of  pigeons,  181 
Chromogenic,  definition  of,  2 
Cladius  stains  for  cover-glass  specimens, 

20 
Cladius  stains  for  cover-glass  sections, 

35 
Cladothrices,  general  characters  of,  4, 

229 

Cladothrix  dichotoma,  229 
intricata,  229 
ochracea,  230 

Classification  of  bacteria,  4,  5 
Clostridium,  7 

butyricum,  221 
Clyers,  103 

Cocci,  general  characters  of,  4 
Coccidia,  254 
in  lambs  and  pheasants,  254 

cattle,  254 

elimeria  falciformis,  255 
Coccidium  oviforme,  254 

perforans,  254 
Colon  bacillus,  174,  176 

Cesaris-Demel's     differentiation 

method,  175 

differential  diagnosis  table,  175 
Piorkowski's  differentiation  method, 

175 
Kashida's    differentiation    method, 

176 

Comma  bacillus  of  Koch,  153,  158 
Contact  specimens,  preparation  of,  18 
Corn-stalk  disease,  200 

S  2 


278 


INDEX 


Cover-glass  specimens,  preparation   of, 

15,  16,  IT,  18 
examination,  18 

method  of  re-mounting  and  re-stain- 
ing, 29 

necessary  precautions  with,  29 
Loffler's  method  of  cleansing,  144 
Kischensky's  quick  method  for,  271 
Semenowicz     and     Marzinowsky's 

special  method  for,  271 
Cryptococcus  of  Rivolta,  135 
Culture  media,  preparation  of,  47-68 
Cultures,  method  of  obtaining  pure,  from 

plates,  63 

Culture  methods  with  water  of  conden- 
sation, 60 
method  of  detecting  indol  and  nitrates 

in,  72 
preparation  of,  from  organs,  tissues, 

etc.,  79 

Cygnaeus's  experiments  with  B.  typhi 
abdominalis,  173 

D 

Demel,  Cesaris-,  differentiation  of  colon 

bacillus,  175 
Deneke's  vibrio,  159 
Differentiating  agents,  45 
Dimensions  of  bacteria,  5 
Diphtheria,  bacillus  of,  162-167 

differential  diagnosis  of,  165 
staining,  method  of  Neisser,  23 

immunity,  165 

Roux's     method     of     immunizing 
horses,  166 

antitoxin,  preparation  of,  165,  166 
Schering's,  167 

experiments  of  Schotellius,  164 
Diphtheria  of  calves,  168,  169 

chickens,  167,  168 

pigeons,  167,  168 

Diplococci,  general  characters  of,  4 
Diplococcus  of  pneumonia,  91 

pleuro-pneumonia  in  horses,  99 
Discomyces,  100 
Disinfecting  solutions,  44 
Drumstick  bacteria,  7,  227 
Duck  cholera,  181,  182 
Dunham's  peptone  media,  51 
Dysenteria,    haemorrhagica    coccidiosa, 

254 

E 

Earth,  examination  of,  84 
Fraenkel's  method,  84 
principal  bacteria  found  in,  84 
examination      of,      Winogradsky's 
method,  85 

Eczema  epizootica,  203-204 

Egg  media,  51,  52 
Hueppe's  method,  51 
Giinther's  do.,  52 
albumen,  52 


Ehrlich's  anilin  water  stains,  42 

stain  for  tubercle  bacilli  in  sections,  36 

in  cover-glass  specimens,  21 
Eisner's     method     for     demonstrating 

typhoid  bacilli,  83 

Embedding  in  celloidin,  method  of,  31 
Endocarditis  verucosa  bacillosa,  194 
Endospore  formation,  6 
Enteritidis,  bacillus  of,  125,  126 

symptoms  in  man,  126 
Equi-intestinalis,  bacillus  of,  176 
Equine  tuberculosis,  144,  145 

abdominal  form,  144 

thoracic  do.,  144 

distinctive  features  of,  144,  145 
Erysipelas,  streptococcus  of,  90 
Esmarch's  roll  culture  method,  61 


Farcy,  African,  135 

Farcy  in  cattle,  108 

Favus,  235 

Fermentation,  production  of,  70 

Finkler  and  Prior's  vibrio,  158 

Flagella,  5 

Bowhill's  method  of  staining,  26,  27 

Loffler's  do.,  26 
Foot  and  mouth   disease,  bacteria  in, 

203,  204 
Fowl  cholera,  182,  183 

immunity,  183 

Fraenkel's    anaerobic    culture    method, 
65 

examination  of  earth,  do. ,  84 

diplococcus  of  pneumonia,  91 
Freezing,  preparation  of  tissues  for,  31, 

32 

Friedlander's  bacillus  of  pneumonia,  92 
Frog  spawn  fungus,  223 
Fungi,  Unna's  method  of  staining,  23 

media  for,  57 

Sabouraud's  do.,  57 
Fusisporium  moschatum,  242 


Gabbet's  stain  for  tubercle  bacilli,  41 
Gas  in  cultures,  nature  of,  70,  71 

composition  of,  71,  115 

quantitative  relations  of,  71 

explosive  nature  of,  71,  72 
Gastromycosis  ovis,  271 
Gelatine  media,  52,  53 
Glanders,  bacillus  of,  129-136 

staining  of,  22,  37,  130 

bacteriological  diagnosis  of,  133 

Strauss'  method  for  do.,  133 

mode  of  infection,  132 

Prieur  on  do. ,  132 

Nocard  on  do.,  132 

Schiitz  on  do.,  132 

mallein,  133 

Hunting  on,  134 
Nocard  on,  136 


INDEX 


279 


Glycerine  media,  48,  54 
Gonococcus,  94-96 

bacteriological  diagnosis  of,  95,  96 

stain  for,  22 

Knack's  do.,  23 
Gram's  method,  19 

Giinther's  modification  of,  34,  35 

bacteria  stained  by,  19,  20 

control  test,  19 

mordant,  43 

Green  pus,  bacillus  of,  96 
Grouse  disease,  184 
Gruber's  reaction  in  Asiatic  cholera,  155 

typhoid  fever,  184 
Giinther-Gram  modification,  34,  35 

egg  medium,  52 

H 

Haffkine,  anti-plague  serum,  199,  200 
Hanging-drop,  preparation  of,  16 

method  of  examining,  16 

Nuttall's  do.,  16 
Hay  bacillus,  210 
Herpes  tonsurans,  236 
Hesse's  method  for  anaerobic  cultures,  65 

air  examination,  80 
Holz's  potato  gelatine,  50,  171 
Hot-air  oven,  description  of,  7,  8 
Hydrogen   in  anaerobic  cultures,    test 

for,  66 
Hydrophobia,  blastomycetes  in,  252 

inoculation  of  rabbits  for  diagnosis  of, 

77 
Hyphomycetes,  general  characters  of,  1 

or  mould  fungi,  235 

I 

Ictero-haemoglobinuria  of  sheep,  265 
Ilosvay,    Griess's   method  of  detecting 

nitrites,  72 
Immunity  in  malignant  oedema,  114 

tetanus,  119,  120 

tuberculosis,  147,  148 

botulismus,  125 

diphtheria,  165 

influenza,  179 

swine  fever,  191 

fowl  cholera,  183 

bubonic  plague,  199 

swine  erysipelas,  194 

southern  fever  of  cattle,  264 
Incubator,  description  of,  68 
Indol  in  cultures,  detection  of,  72 
Infectious  broncho-pneumonia,  200 
Inflammation,  bacteria  found  in,  87 
Influenza,  bacillus  of,  176-179 

Pfeiffer  method  for  pure  cultures  of, 
177 

immunity  in,  179 
Inoculation  of  animals,  74-77 

rabbits  for  diagnosis  of  rabies,  77 

observation  of  animals  after,  77,  78 


Inoculation  of  animals,  syringes  for,  75 
into  the  eye,  76 

circulation,  75 

peritoneal  cavity,  75,  76 
Involution  forms,  4,  5 
lodococcus  vaginatus,  205 
Ixodes  bovis,  261,  263 
Ixodic  anaemia  in  cattle,  261 


Johne's    method    of    staining    capsule 

bacteria,  25 
Johnston  Wyatt  or  Widal's  reaction,  172 

K 

KASHIDA,  differentiation  of  the  colon  and 

typhoid  bacillus,  176 
Kasparee's  anaerobic  culture  method,  67 
Kipp's  hydrogen  apparatus,  67 
Kischensky's  method  of  staining  bac- 
teria, 271 

Kitasato,  anaerobic  culture  flask,  66 
Klebs-Loffler  bacillus  of  diphtheria,  162 
Klossia  soror,  265 
Knack's  method  of  staining  gonococci, 

23 

Koch's  steam  sterilizer,  8 
plate  culture  method,  58,  59 

apparatus,  59 

apparatus  for  blood  serum,  56 
anaerobic  culture  method,  64 
safety  burner,  58 
inoculating  needle,  74 

syringe,  75 
method  for  diagnosis  of  cholera  bacilli 

in  water,  83,  157 
original  tuberculin,  145 
new  tuberculin,  146 
Kuhne's  methylene  blue  stain,  38 


LEPROSY,  bacillus  of,  152 
Baumgarten's    method    for    staining-, 

137 

Leptothrix,  definition  of,  4 
buccalis  innominata,  205 
maxima,  205 
maximus,  205 
gigantica,  205 
Leuconostoc  mesenteroides,  223,  224 

indicum,  224 

Liborious's  method  for  anaerobic  cul- 
tures, 66 
tube,  65 

Litmus  tincture,  69 
Lockjaw,  116 

Loffler's  blood  serum  for  diphtheria,  56 
method  for  cleansing  cover-glasses,  44 
staining  flagella,  26 
glanders,  22,  37 
methylene  blue  stain,  41 
mordant,  43 
universal  staining  method,  36 


280 


INDEX 


Lorenz's  vaccine  for  rouget,  195 
Lustgarten's  bacillus  of  syphilis,  137 
Lymphangitis  epizootic,  135 

M 
Malaria,  255 

quartan  parasite  of,  256 
tertian  do.,  256 
quotidian  do.,  256 
malignant  tertian  do.,  257 

mixed  infection,  258 
dry  specimens,  preparation  of,  259 
Plehn's  method  of  staining  parasite 

of,  260 

mode  of  infection,  260 
bovine,  265 

Malassez  and  Vignal's  pseudo-tubercu- 
losis, 150-151 
blue  stain,  150 

Malignant  oedema,  bacillus  of,  113-114 
immunity  in,  114 
in  man,  114 

differential  diagnosis  table,  122 
Malignant  pustule,  111 
Mallein,  diagnosis  of  glanders  with,  134 
preparation  of,  133 
Hunting  on,  134 
Nocard  on,  134 
dose  of,  133 

Mammitis,  cows,  streptococcus  of,  98 
gangrenous  in  ewes,  micrococcus  of,  99 
tubercular,  141 
Mastitis,  see  mammitis,  98 
Meat,  tuberculous,  141 

poisoning,  bacteria  of,  122,  125,  126, 

127,  129 

symptoms  of,  122,  123,  126 
Media,  47-58 
bouillon,  47 

clearing  and  mounting,  46 
grape  sugar,  47 
milk  do.,  48 
glycerine,  48 
potato,  48,  49,  50 
milk,  51 
egg,  51,  52 
peptone  water,  Koch's,  50 

Dunham's,  51 
rosolic  acid,  51 
gelatine,  52,  53 
agar-agar,  52,  54,  55 
blood  serum,  55,  56 
for  mould  fungi,  57 
Sabouraud's,  57 
Winogradsky's,  57 
preparation  of  tubes,  flasks,  etc.,  for, 

57,  58 

bread  paste,  57 

Micrococci,  general  characters  of,  4,  5 
Micrococcus  acidi  lactici,  213 

liquefaciens,  214 
agilis,  211 
aquatilis,  211 
ascoformans,  211 


Micrococcus  auranticus,  210 

candicans,  211 

concentricus,  211 

cremoides,  211 

of  gangrenous  mammitis  in  sheep,  99 

of  gonorrhoea,  94 

prodigiosus,  208,  209 

radiatus,  212 

rossetaceus,  211 

tetragenus,  93 

ureae,  205,  206 

versicolor,  211 
Microscope  of  Zeiss,  15 

Leitz,  15 
Microsporon  furfur,  240 

minutissimum,  240 
Microtome,  Schanze,  33 

Jung,  32 
Milk  bacteria,  213 

method  of  examination  for,  24 

sugar  bouillon,  48 

gelatine,  53 

media,  51 

tubercle  bacilli  in,  141 

special  bacillus  isolated  from,  270 
Milzbrand,  109 
Monas  prodigiosa,  208 
Morbificans  bovis,  126,  127 

action  on  man,  127 
Mordants,  43 

Morphology  of  bacteria,  4,  5 
Mould  fungi,  1. 

Mouse  septicaemia,  bacillus  of,  195 
Mouth,  bacteria  found  in,  205 
Mucors,     characters     of,     corymbifer, 
mucedo,    pusillus,    ramosus,    race- 
mosus,   stolonifer,  rhizopodiformis, 
241 
Mycoderma  aceti,  220 

Pasteurianum,  220,  221 

Cerevisiae  et  vini,  249 
Mykodermoid,  '  Johne,'  100 

N 

Nasal  catarrh,  92 

Neisser's  stain  for  diphtheria  bacilli,  23, 
42 

gonococcus,  94 
Nephritis,  bacteria  in,  206 
Nitrates,  233 
Nitrifying  bacillus  of  Winogradsky,  233 

bacteria,  232 
Nitrites,  Ilosvay's  and  Griess's  test  for, 

72 

Nitro-bacteria,  232 
Nitroso-bacteria,  232 

coccus,  233 
Nitroso-monas,  233 
Nitroso-indol     reaction    in     cholera 

Asiatica,  155 

Nutrient  gelatine,  preparation  of,  52 
Nuttall's  steam  sterilizer,  11 

microscopic  thermostat,  17 

platinum  culture  spear,  79 


INDEX 


281 


Nuttall's  experiments  with  sterile  food, 

176 

bacillus  of  bubonic  plague,  198 
bacillus  anthracis,  112 


Oidium  albicans,  237 

lactis,  238 

Onychomycosis,  235 
Orchitis  caused  by  bacillus  mallei,  133 

orchiticus,  135 
Otitis  media  acuta,  92,  179 
Overlack's  inoculation  syringe,  75 


Paramaecium  coli,  254 
Parrots,  disease  of,  91 
Pasteur's  anthrax  vaccine,  111 
chicken  cholera  do.,  183 
rouget  do.,  194 

Pathogenic  blastomycetes,  254 
Peckham's  indol  reaction  with  bacillus 

typhi  abdominalis,  171 
Penicillium  glaucum,  238 
Peptone  rosolic  acid  media,  51 
water  do. ,  '  Dunham,'  51 

« Koch,'  50 
Petri-dishes,  sterilization  of,  12 

method  for  examination  of  air,  86 
Pfeiffer's  method  for  pure  cultures  of 

influenza,  177 

reaction  for  B.  of  Asiatic  cholera,  155 
Pheasant  disease,  183 
Phosphorescent  bacteria,  225,  226 
Photo-bacteria,  225,  226 
Photogenic,  definition  of,  2 
Photomicrography,  265 
Piorkowski's  differentiation  between  the 

typhoid  and  colon  bacilli,  175 
Plasmodia,  3 
Plasmodium  malarias,  255 

Plehn's  method  of  staining,  260 
Plate  cultures,  methods  for  hard  sub- 
stances, favus,  etc.,  60 
Koch's  original  method,  58 
quantitative  method,  64 
Plehn's  method  of  staining  plasmodium 

malarise,  260 
Pleomorphism,  5 
Pleuro-pneumonia  contagiosa  bovis,  201- 

203 

Nocard's  investigations,  202 
experimental  inoculation,  203 
Pneumo-enteritis  of  sheep,  186 

swine,  188 

Pneumonia,  bacillus  of  Friedlander,  92 
broncho-infectious  bovis,  200 
diplococcus  of  Fraenkel,  91 
infectious,  of  pigs,  192 
pleuro-contagiosa,  horse,  99,  100 
bovis,  201 


Pneumonia,  in  swine  fever,  190,  191 

of  turkeys,  185,  186 
Post-mortem     examination     of     inocu-' 

lated  animals,  78 
Potato  bacillus,  210 
culture  media,  48 

Esmarch's  method,  49 
Holz's  do. ,  50 
Roux  and  Globig's,  do.,  49 
water,  50 

Proteus  fluorescens,  128 
in  chicken  disease, 
mirabilis,  127,  129 
vulgaris,  bacillus  of,  127 

bacteriological  diagnosis,  129 
in  cholera  infantum,  129 
putrid  intoxication  with,  128 
swarming  islands,  128 
symptoms  in  man,  129 
Zenkeri,  127,  129 
Protozoa,  3,  253 

Pseudo-diphtheria,  bacillus  of,  165 
differentiation,  165 
influenza,  bacillus  of,  179 
Pseudopodia,  3 
Pseudo-tuberculosis,  bacillus  of,  108, 109, 

140,  149,  151 

animal  parasites,  cause  of,  150 
aspergillus,  glaucus,  »nd  fumigatu 

cause  of,  151 
in  calves,  150,  151 
causal  factors  of,  150 
in  cats,  150 
Courmont's,  151 
in  dogs,  150 
man,  150 

Malassez  and  Vignal's,  150 
in  pigeons,  151 

sheep,  150 

Pseudo-typhoid  bacilli,  207 
Pus,  blue  and  green,  bacillus  of,  96 
Pustula  maligna,  111 
Pyocyanin,  96 
Pyogenic,  definition  of,  2 
Pyrosoma  bigeminum,  261 

R 

RABIES,  diagnosis  of,  77 
Ragpickers'  disease,  111 
Rauschbrand  bacillus,  114 
Reagents,  differentiating,  45 

solids,  45 

liquids,  45 

special  actions  of,  45 
Red  dysentery  of  cattle,  254 
Reichel's  filter  for  bacteria,  73 
Relapsing  fever,  spirillum  of,  160,  161 
Rhinitis  fibrinosa,  166 
Rhizobium  leguminosarum,  232 
Rinder-seuche,  bacillus  of, 
Roll  cultures,  61,  62 
Root  bacillus,  209 
Rosolic  acid,  51,  70 


.282 


INDEX 


Roth's    method  for   tubercle  bacilli  in 

butter,  25 

Rotz  bacillus,  129-134 
Rouget,  bacillus  of,  193,  195 
Roux  on  cerebral  tetanus,  121 
Roux's  double  stain,  41 

method  of  immunizing  horses,  166 


Sabouraud's  medium  for  favus,  57 
Saccharomyces  cerevisiae,  I.,  244 

ascospores  of,  244 
ellipsoideus,  I.,  245 

II.,  246 

Pastorianus,  I.,  256 
II.,  24T 
III.,  247 
apiculatus,  248 
anomales,  248 
marxianus,  248 
membranaefaciens,  249 
exiguus,  249 
acidi  lactici,  249 
hominis,  250 
litogenes,  251 
neoformans,  251 
subcutaneus  tumefaciens,  251 
in  hydrophobia,  252 
Saliva,  bacteria  in,  187,  205 
Sanarelli's  experiments  with   B.    typhi 

abdominalis,  173 
Saprogenic,  definition  of,  2 
Saprophyte,  definition  of,  2 
Sarcinae,  characters  of,  4 
yellow,  211 
white,  211 
red,  211 
orange,  211 

Schering's  diphtheria  antitoxin,  167 
Schiller's  experiments  with  tuberculosis, 

139 

Schizomycetes,  characters  of,  1 
Schotellius'  experiments  with  diphtheria, 

164 
Schiitz,  diplococcus  of  pneumonia,  horse, 

99,  100 

streptococcus  of  strangles,  97 
tinea  galli,  236 

Schweine-seuche,  bacillus  of,  192 
differential  diagnosis  table,  196 
Schweine-rothlauf;  193 
Schweinitz,   immunity    in    hog  cholera 

and  schweine-seuche,  191 
Sections,  common  faults  in  staining  of, 

39 

cutting  of,  33 
Cladius  stain  for,  35 
Ehrlich's  stain  for  tubercle  bacilli  in, 

36 

examination  of,  for  bacteria,  31 
Gram-Gunther  stain  for,  34,  35 
precautions  with,  39 
staining  of,  for  bacteria,  33 


Sections,  Unna's  dry  method,  38 

Weigert's  stain  for,  34 
Septicaemia  haemorrhagica,  bacteria  of, 

179-201 

Smegma  bacillus,  137,  152,  153 
Smith's  method  for  isolating  intestinal 

bacteria,  84 
fermentation  tube,  70 
.  Soil  bacteria,  206 
Solid  culture  media,  55 
Solution  for  cleansing  and  disinfecting,  44 
Southern  cattle  fever,  261 
parasite  of,  261 
anatomical  changes  in,  262 
mode  of  infection,  263 
ticks  in,  263 
immunity  in,  264 
Koch's  experiments  with,  264 
Sphaerococcus  acidi  lactici,  214 
Spirilla,  general  characters  of,  4 
Spirillum  dentium,  205 

of  Finckler  and  Prior,  158,  159 

Miller's,  160 

Obermeiri,  160,  161 

of  relapsing  fever,  160,  161 

sputigenum,  205 

tyrogenum,  159 

undula,  162 

majus,  and  minus,  162 
Spirochaete,  characters  of,  4 

Obermeiri,  160,  161 
Spores,  endogenous,  6 
formation  of,  6,  7 
vitality  of,  6 

ordinary  method  of  staining,  27 
special  do.,  anthrax,  28 
Stains,  Bowhill's  orcein  for  flagella  and 

bacteria,  43 

chloroform  fuchsin  solution,  42 
Cladius's,  for  cover-glass  specimens,  20 
for  cover-glass  specimens  and  sections, 

41 

for  daily  use,  40 
Ehrlich's  anilin  water,  42 
fuchsin,  42 
gentian  violet,  42 
methyl  violet,  42 
Gabbet's  solution,  41 
Gram's,  for  cover-glass  specimens,  19 
Kuhne's  methylene  blue,  38,  42 
Loffler's  do.,  41 
Malassez's  blue,  150 
Neisser's,  42 
Roux's  double,  41 
stock  solutions,  40 
Staphylococci,  characters  of,  4 
Staphylococcus  cereus  albus,  88 
flavus,  88 
griseus,  88 
pyogenes  albus,  88 
aureus,  87 
citreus,  88 
rosaceus,  88 
differential  table,  88 


INDEX 


283 


Steam  sterilizer,  Arnold's,  8 
Bowhill's,  9 
Koch's,  8 
Novy's,  8 
Nuttall's,  8 

Sterilization  by  heat,  7,  8 
Tyndall's  discontinuous  method,  11,  12 
of  post-mortem  instruments,  12 
Sterilizer,  hot-air,  8 
Strangles,  differential  diagnosis  of,  98 

streptococcus  of,  in  horse,  97 
Strauss'     method     for     diagnosis     of 

glanders,  133 

Streptococci,  characters,  4,  5 
Streptococcus  acidi  lactici.  214 
of  erysipelas,  90 
Hollandicus,  214,  239 
lanceolatus  Pasteur,  91 
of  mammitis  in  cows,  98,  99 
perniciosus  psittacorum,  91 
pyogenes,  89 

of  strangles  in  horses,  Schiitz,  97 
Streptothrix,  characters  of,  4,  102 
actinomyces  bovis,  102 
hominis,  106 

differential  diagnosis  of,  107 
Eppinger,  108 
Farcinica,  108 
Hoffmann,  106 
madurae,  107 

Suppuration,  bacteria  found  in,  87 
Swine  erysipelas,  193-195 

differential  diagnosis  table,  196 
immunity,  194,  195 
Lorenz's  vaccine,  195 
Pasteur's  do.,  194 
post-mortem  appearances,  194 
symptoms  of,  193 
Swine  fever,  bacillus  of,  188-191 
differential  diagnosis  table,  196 
flagella,  188 
identity  of  English  and  American, 

188 

immunity  in,  191 
lesions  of,  189,  190,  191 
pneumonia  in,  190,  191 
symptoms  of,  189 
Swine  plague,  Billings,  188 
Swine  tuberculosis,  145 

mode  of  infection,  145 
Symptomatic  anthrax,  bacillus  of,  114- 

116 

characteristic  lesions,  114,  115 
differential  diagnosis  of,  115,  116 

table,  122 
vaccination,  116 
French  method,  116 
Kitt's  do.,  116 
Syringes  for  inoculation  of  animals,  75 


Tabes  mesenterica,  141 
Technique,  principles  of,  13 


Tetanus,  bacillus  of,  116,  117 
flagella,  117 
immunity  and  anti-tetanic  serum,  119- 

121 

motility,  117 
staining  reactions,  117 
cerebral,  121 

differential  diagnosis  table,  122 
immunizing  of  horses,  119,  120 
Kitasato's  method  for  pure  cultures, 

117 

Kitt's  do.,  118 
symptoms  of,  121 
Bacelli's  treatment  of,  273 
Tetrads,  definition  of,  4 
Texas  cattle  fever,  261 
Thermophilic  bacteria,  226 
Thermostat,  microscopic,  '  Nuttall's,'  17 
Thierfelder  and  Nuttall,  feeding  animals 

with  sterilized  food,  176 
Thiogenic,  definition  of,  2 
Thrush,  237 
Tinea  galli,  236 
Torulae,  characters  of,  250 

white,  rose,  and  black,  250 
Trichophyton  tonsurans,  236 

microsporon  and  megalosporon,  237 
Trommelschlager,  bacilli,  227 
Tuberculin,  dose,  146 

Koch's  new  preparation  of,  145 

original  do.,  146 
method  of  inoculation,  146 
precautions  before  and  after  use,  146, 

147 
Trudeau   and   Baldwin's  experiments 

with  '  New,'  145 
Tuberculosis,  bacillus  of,  136 
in  butter,  141,  142 
differential  staining,  137 
Ehrlich's  method  of  staining,  21,  22,  36 
immunity,  147 

Koch's  method  for  pure  cultures,  137 
in  meat,  141 

micro-mechanical  reactions,  136 
in  milk,  141 
staining  reactions,  136 
Herman  and  Morgenroth's  method  of 

demonstrating,  in  butter,  143 
Schiller's     experiments     with      dead 

bacilli,  139,  140 

Ziehl-Gabbet  method  of  staining,  21 
avian,  148 
bovine,  140 
canine,  145 
equine,  144 
of  swine,  145 

Turkeys,  disease  of,  185,  186 
Typhus  abdominalis,  169-174 

Eisner's  method  for  detection  in  water, 

83 

Typhi  murium,  187,  188 
Typhoid  fever,  bacillus  of,  169-176 
differential  diagnosis  table,  175 
flagella,  169 


INDEX 


Typhoid    fever,   bacillus   of,   involution 
forms,  169 

Cygnaeus'  experiments  with,  173 

Sanarelli'sdo.,  173 

Gruber's  reaction,  171 

specific  do.,  171,  172 

Widal's  do.,  171,  172 

Wyatt  Johnson  on,  173 
Typhoid  of  mice,  187,  188 


U 


UNNA,  stain  for  fungi,  23 

dry  method  for  sections,  38 
Urethritis  catarrhalis,  96 
Urobacillus  Pasteuri,  206 


VACCINATION  in  anthrax,  111 

symptomatic  do.,  116 

rouget,  194,  195 

swine  fever,  191 

chicken  cholera,  183 
Van  Ermengen,  botulismus,  132 
Vibrio,  characters  of,  4 

aquatilis,  212 

Berolinensis,  155,  212 

cholerae  Asiaticse,  153,  158 
in  water,  83 

Deneke,  159 

Dunbar,  213 

Gindha,  212 

Lissabon,  213 

Massauah,  213 

Metschnikoff,  155,  212 

phosphorescens,  Dunbar,  213 

proteus,  158 

rugula,  161 


W 

WATER,  bacteria  in,  206,  213 
method  of  examination,  81,  82 
qualitative  method,  83 
Eisner's  method,  Bac.  typhi  abdomin- 

alis,  83 
Koch's    method    for    vibrio    cholerae 

Asiaticae,  83,  157 
Smith's  method  for  isolating  intestinal 

bacteria,  84 

quick  method  for  pathogenic  germs,  83 
transportation  case  for  analyses,  82 
Weigert's  stain  for  fibrin  or  bacteria,  37 

sections,  34 
Weil's  disease,  128 
White  diarrhoea  in  calves,  180,  181 
Widal's  reaction  for  typhoid  fever,  171 
Winogradsky's  method  for  examination 

of  earth,  85 
medium,  57 

Wolffhugel's  counting  apparatus,  63 
Woodhead's  experiments  with  Bac.  pyo- 

cyanus,  97 

Woolsorters'  disease,  111 
Wurtz's  lactose  litmus  agar,  54 
Wurzel  bacillus,  209 


YEAST  fungi,  1,  243 
pure  cultures  of,  243 


ZIEHI>GABBET  stain  for  bacillus  tubercu- 
losis, 21 

Zoogloea,  definition  of,  4 
Zoogloeic  tuberculosis,  150 
Zymogenic,  definition  of,  2 


OLIVER    AM)    liOVl),    PRINTER 


DATE  DUE  SLIP 

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STAMPED  BELOW 


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JUL  15  1940 

MAR  3  0  tQ«?n 


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